Characteristics, and Comparisons with the Eucaryotic Process

Transcription

1 MICROBIOLOGICAL REVIEWS, Sept. 1989, p Vol. 53, No /89/ $02.00/0 Copyright 1989, American Society for Microbiology Insertion of Proteins into Bacterial Membranes: Mechanism, Characteristics, and Comparisons with the Eucaryotic Process MILTON H. SAIER, JR.,'* PAMELA K. WERNER,' AND MATTHIAS MULLER2 Department of Biology, C-016, University of California San Diego, La Jolla, California 92093,1 and Biochemisches Institut, Universitat Freiburg, D-7800 Freiburg, Federal Republic of Germany2 I. INTRODUCTION II. TARGETING TO THE ENDOPLASMIC RETICULUM OF EUCARYOTIC CELLS: CHARACTERISTICS OF THE PROTEIN INSERTION-SECRETION SYSTEM A. Involvement of the SRP B. The SRP Receptor and Ribophorins C. Signal Sequence Receptor in the Endoplasmic Reticular Membrane III. INSERTION OF INTEGRAL MEMBRANE PROTEINS INTO THE ENDOPLASMIC RETICULUM IV. TARGETING TO THE ENVELOPE FRACTION OF BACTERIA: CONSTITUENTS OF THE PROTEIN INSERTION-SECRETION SYSTEM...I A. Probable Antifolding Receptor Proteins: the SecB Protein, Trigger Factor, and the Heat Shock GroEL Protein of E. coli B. The SecA Protein C. Soluble Export Factors from E. coli D. The SecY Protein E. Other Possible Components of the E. coli Export System Defined by Genetic Analyses F. Comparison of the Insertion Machinery of Gram-Positive Bacteria with That of E. coli G. Bacterial RNA Species Showing Homology with Eucaryotic SRP RNA V. INSERTION OF INTEGRAL MEMBRANE PROTEINS INTO THE CYTOPLASMIC MEMBRANE OF E. COLI A. Insertion of Small versus Large Membrane Proteins B. Charge Distribution as a Determinant of Integral Membrane Protein Topology C. In Vitro Membrane Insertion of the E. coli Lactose Permease VI. TEMPORAL MODE OF PROTEIN TRANSPORT ACROSS CELLULAR MEMBRANES VII. DISTINCTIVE FEATURES OF MITOCHONDRIAL TARGETING A. N-Terminal Amphipathic Targeting Sequences B. Physicochemical Properties of Mitochondrial Targeting Peptides C. Targeting Information Encoded within the Primary Sequences of Mitochondrial Precursor Proteins VIII. DISTINCTIVE FEATURES OF BACTERIAL PTS PERMEASE INSERTION A. N-Terminal, Amphipathic Leader Sequences B. Statistical and Functional Analyses of Proteins with N-Terminal, Amphipathic, Potentially oa-helical Sequences C. Physicochemical Properties of PTS Targeting Peptides and Comparison with Mitochondrial Targeting Peptides D. Targeting Conclusions Resulting from Studies with Fusion Proteins E. Internal Amphipathic a-helices as Potential Translocation Signals IX. UNIFIED MODEL OF PROTEIN INSERTION ACKNOWLEDGMENTS ADDENDUM IN PROOF LITERATURE CITED "The basic life-endowing molecular processes had to exist prior I. INTRODUCTION to extensive evolutionary divergence. Thus, we should expect that these processes are governed by the same principles, and that even Biochemical and genetic studies have revealed that prothe molecular details will sometimes prove to be conserved through- tein targeting and transmembrane transport in the eucaryotic out evolutionary history." endoplasmic reticulum are strikingly similar to these pro- M. H. Saier and G. R. Jacobson cesses in the gram-negative bacterium Escherichia coli. Both The Molecular Basis of Sex and Differentiation, 1984 processes occur by apparently homologous mechanisms, with conservation of many specific functions. For example, the signal sequences (also referred to as signals, presequences, leader sequences, and leaders) and stop-transfer sequences of transmembrane and secretory protein precur- * Corresponding author. sors of eucaryotes and bacteria are structurally and function- 333

2 334 SAIER ET AL. ally similar (see sections III and V). Cleavage of aminoterminal signal sequences by signal peptidases occurs with similar specificities (89, 184, 220, 322); the transmembrane translocation process is dependent on energy, adenosine triphosphate (ATP), and, sometimes, the electrochemical membrane potential (44, 101, 110; P. C. Tai, in T. A. Krulwich, ed., Bacterial Energetics, in press), and both systems require a multicomponent export-insertion enzyme complex (190, 255, 260, 317). Biochemical studies of the mammalian endoplasmic reticular secretory system have led to the identification of five functionally distinct, complex components of the export machinery (see section II). First, a cytoplasmic signal recognition particle (SRP) binds the hydrophobic signal sequence of an unprocessed secretory protein, regulates the rate of translational elongation, and initiates translocation of the growing polypeptide chain across the membrane. Second, an SRP receptor (the docking protein) in the endoplasmic reticular membrane serves to associate the SRP-polysome-nascent polypeptide complex with the membrane and the translocation machinery and then to allow release of SRP (317). Third, an integral membrane protein, the signal sequence receptor, appears to interact with the signal sequence once it has been delivered to the membrane by the SRP complex (330). Fourth, the ribosome binds to ribosome receptor proteins which are believed to be the two rough endoplasmic reticulum-specific glycoproteins called the ribophorins (56, 112). Finally, following translocation of the protein across the membrane, the signal peptidase cleaves the signal peptide from the remainder of the protein, yielding the mature form. This last-mentioned enzyme has been purified and shown to consist of six distinct polypeptide chains (86). Corresponding studies with E. coli have allowed the identification of soluble proteins which maintain precursor proteins in appropriately unfolded states. Known proteins with this function include the SecB protein, the GroEL protein, and trigger factor (see section IV.A). Additionally, biochemical studies show that a cytoplasmic, SRP-like protein particle, as well as some smaller proteins, has been implicated in the translocation process (see section IV.C), and genetic studies show that both a peripheral membrane protein (SecA) and an integral membrane protein (SecY) are essential to the insertion-secretion process (see sections IV.B and IV.D). Genetic evidence for the presence of other constituents of the system has been presented (see section IV.E). In Bacillus subtilis and Staphylococcus aureus, a four-constituent-protein complex appears to serve an SRPlike function (see section IV.F). Bacterial ribonucleic acids (RNAs) homologous with the SRP RNA have been identified in both gram-positive and gram-negative eubacteria as well as archaebacteria (see section IV.G). Import of proteins into mitochondria appears to resemble targeting and secretion of proteins across the endoplasmic reticular membrane in some mechanistically important respects (see section VII). However, the N-terminal targeting sequences of nucleus-encoded mitochondrial proteins are amphiphilic (amphipathic) structures rather than hydrophobic sequences. Recently, similar structures have been found to be capable of targeting bacterial proteins to the envelope fraction of E. coli and initiating their insertion into, or translocation across, the procaryotic cytoplasmic membrane (see section VIII). Although proteins are generally translocated across the endoplasmic reticular membrane cotranslationally and imported into mitochondria posttranslationally, both mechanisms appear to be operative in bacteria (see MICROBIOL. REV. section VI). These results suggest additional mechanistic parallels between the procaryotic and eucaryotic translocation systems (see section IX). The present review serves to analyze the bacterial insertion process in detail and to draw parallels with the eucaryotic process when warranted. We do not intend to provide comprehensive coverage of the subject but, rather, to emphasize recent investigations which we believe to provide definitive information about the insertion process. Thus, the protein components of the secretion-insertion machinery are analyzed, but the enzymes responsible for cleavage of the signal peptides from the mature forms of secreted proteins following their transmembrane translocation are not. The cleavage enzymes are clearly nonessential to the export of secretory proteins and the insertion of integral proteins into biological membranes (see, for example, references 189, 249, 339, and 340). Because protein secretion across the outer membrane of gram-negative bacteria appears to occur by distinct processes involving independently functioning enzyme complexes which have nothing to do with the process of protein insertion into the inner membrane ( ), this subject is not covered here. Furthermore, of the eucaryotic translocation systems studied, the endoplasmic reticular system is emphasized, because of the relatively advanced state of our knowledge regarding its enzymology. The mitochondrial import system is considered from a comparative standpoint, with emphasis on its distinctive features. Targeting of endoplasmic reticular proteins to specific compartments (the endoplasmic reticulum, the Golgi apparatus, lysosomes, and the plasma membrane or the extracellular fluid) and targeting of cytoplasmic enzymes to other organelles (peroxisomes, chloroplasts, and nuclei) are not considered. The sorting process in polarized cells (107, 227, 228) is also beyond the scope of this review. With the exception of signal peptide cleavage, most aspects of bacterial targeting and secretion are discussed. Excellent recent reviews have appeared concerning export to and from the endoplasmic reticulum (36, 69, 185, 235, 259, 276, 309, 327, 344), mitochondrial targeting (204, 207, 225, 258, 299, 300), chloroplast targeting (138, 266, 309), targeting to other organelles (108, 136, 140, 155, 188, 197, 209, 303, 309), and bacterial secretion (20, 36, 98, 139, 158, 190, 192, 216, 221, 229, 288, 327). In addition, several aspects of protein secretion and export in bacteria have been discussed (295, 335). II. TARGETING TO THE ENDOPLASMIC RETICULUM OF EUCARYOTIC CELLS: CHARACTERISTICS OF THE PROTEIN INSERTION-SECRETION SYSTEM A characteristic feature of secretory proteins targeted to the endoplasmic reticulum of a eucaryotic cell is the N- terminal signal sequence of 15 to 30 residues (23). These sequences consist of a positively charged N terminus, a central hydrophobic region, and a C-terminal region predominating in polar residues (304). Most signal sequences are proteolytically removed by a specific peptidase during transport into the endoplasmic reticular lumen. Tabulation of many signal sequences and analyses of their characteristics have been published by Watson (321) and von Heijne (309). A. Involvement of the SRP The SRP of eucaryotic cells is a ribonucleoprotein complex composed of six distinct polypeptide chains and a 300-nucleotide RNA molecule. The complex mediates targeting of nucleus-encoded proteins to the endoplasmic retic-

3 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 335 (a) ribosome binding (b) signal recognitim (c) elongation anrest (d) SR? receptor recognition (e) nbosome-srp (f) ribosome-membrane dissociation I (g) elongation anest release association (h) transmembrane translocation promotion FIG. 1. Proposed temporal flow chart for the involvement of SRP in the coupled translation-transmembrane translocation of a secretory protein in a eucaryotic cell (see text for an explanation). ulum (for reviews, see references 69, 276, 315, and 317). Much is known about the structure-function relationships of this complex. The results reviewed in this section clearly indicate that in an in vitro-reconstituted system with canine pancreas SRP, SRP links protein synthesis (ribosome-mediated translation) to initiation of the transmembrane protein translocation process, allowing these two conceptually distinct processes to be functionally coupled. Figure 1 provides a proposed temporal flow chart for the secretory events in which SRP participates. SRP first binds a ribosome (a), forming a loose complex (micromolar affinity) which becomes a tight-binding complex (nanomolar affinity) following synthesis and exposure on the ribosomal surface of the signal sequence of the partially translated secretory protein (b). Signal recognition gives rise to translational elongation arrest (c), followed by binding of the translation machinery-srp complex to a specific protein in the membrane, the SRP receptor or docking protein (d), while the signal sequence becomes bound to the signal sequence receptor. This last-mentioned event is accompanied by ribosome-srp dissociation (e), ribosome-membrane association (f), possibly involving one or both of the ribophorins, and release of SRP from the complex. These events, in turn, may allow release from elongation arrest (g) and promotion of transmembrane translocation (h). Although this model portrays cotranslational translocation as the normal secretory pathway, it is clear that posttranslational transmembrane translocation of numerous eucaryotic and procaryotic proteins can also occur (see section VI). Several proteins have been shown to be translocated across the endoplasmic reticular membrane by an SRPindependent mechanism. These proteins include prepromelittin (180) and truncated forms of prelysozyme (129). The common denominator of these examples is an overall length of the primary translation product of less than approximately 70 amino acids. These proteins are likely to be released from the ribosome before being recognized by SRP. This fact is of considerable interest since the only bacterial proteins known to be inserted by a SecA-SecY independent mechanism are the small M13 and PF3 bacteriophage coat proteins (see section V.A). 5' SRP receptor binding and Translocation promotion FIG. 2. Schematic depiction of the structure of the ribonucleoprotein, SRP, which consists of a 300-nucleotide RNA molecule and six constituent proteins (circles). Numbers indicate the molecular masses of the protein subunits in kilodaltons. The primary functions of the various SRP proteins are also indicated. The three-dimensional folding pattern of the SRP RNA is not accurately depicted. As mentioned above, active SRP consists of a single, short RNA molecule to which six distinct protein subunits are noncovalently bound. The constituents, together with some molecular mass and topological information, are shown schematically in Fig. 2. The complex appears to consist of two heterodimeric proteins (9 plus 14 kilodaltons [kda] and 68 plus 72 kda), as well as two interacting but easily dissociable monomeric proteins (19 and 54 kda), which are bound to three distinct sites on the RNA strand and are present in the complex in equimolar amounts. The interactions between the nucleic acid and the protein constituents can be weakened merely by removal of divalent cations with chelating agents (314) or by inclusion of high salt levels (267). The resultant monomeric and dimeric proteins then sediment in a sucrose density gradient differently from the intact ribonucleoprotein complex. Readdition of Mg2+ to a mixture of the separated constituents results in functional reconstitution (272). The polypeptide chains of the two heterodimers have not yet been separated from each other with retention of activity following reconstitution of the complex. In isolated form, the individual proteins exhibit no detectable activity unless the complex is reconstituted. Of the two monomeric constituents of SRP (molecular masses of 19 and 54 kda), the 19-kDa protein, but not the 54-kDa protein, apparently binds directly to the RNA. The sequence of the 19-kDa protein has recently been reported (165). The 54-kDa protein presumably binds to the complex via the 19-kDa protein. On the basis of nuclease digestion experiments of intact SRP followed by sucrose gradient sedimentation analyses (109, 274, 275), as well as footprint analyses of individual reconstitutes (276), the binding sites of the three RNA-binding proteins have been found to be distinct. By using selective dissociation, partial reconstitution, and selective chemical inactivation with, for example, the sulfhydryl reagent N-ethylmaleimide (279), it has been possible to show that the 54-kDa protein is required for signal recognition (Fig. 1, function b), that the 9- plus 14-kDa dimer is essential for elongation arrest (Fig. 1, function c), and that the large protein (68 plus 72 kda) serves to bind the complex to the SRP receptor (Fig. 1, function d) and promote transmembrane translocation (Fig. 1, function h; Fig. 2). The presence of three distinct structural domains in SRP has been confirmed by electron microscopy (11). It should be noted, however, that this modular structure of SRP does not necessarily imply autonomy of the three parts. Assembly of SRP from its constituents exhibits positive cooperativity

4 336 SAIER ET AL. (314), implying the occurrence of long-range interactions between domains. Recently, the interaction of the 54-kDa subunit of SRP with the signal sequence of nascent preprolactin was demonstrated in cross-linking studies. A new method of affinity labeling was used in which lysyl residues, to which a photoreactive reagent was covalently linked, were first incorporated into the signal sequence of preprolactin by means of a modified, charged lysyl-transfer RNA (143, 152, 329). Cross-linking of the nascent preprolactin and the 54-kDa subunit of SRP was then induced by exposure to irradiation. Binding of SRP to preprolactin was shown to be reversible and required the nascent chain to be bound to a functional ribosome (329). The occurrence of SRP-like particles in several nonmammalian eucaryotic organisms has been demonstrated. Translocation-promoting ribonucleoprotein particles have been partially purified from wheat germ (213), maize endosperm (39), and the yeasts Schizosaccharomyces pombe and Yarrowia lipolytica (33, 210, 226). B. The SRP Receptor and Ribophorins The complex of SRP with the ribosome, messenger RNA (mrna), and nascent polypeptide chain binds to the endoplasmic reticulum via an integral membrane protein, the SRP receptor. This protein consists of at least two polypeptide chains, an at-subunit, sometimes called the docking protein (104, 105, 174), and a p-subunit (289). The binding site for SRP is associated with the a-subunit, the primary structure of which has been determined (124, 154). As discussed above, release from translational arrest occurs upon (or shortly after) binding of the SRP-polysome complex to the SRP receptor in the endoplasmic reticulum. Release is probably provoked by an SRP receptor-induced displacement of SRP from the ribosome. In addition, it seems likely that the SRP receptor plays a role in translocation, since a subparticle of SRP which is inactive in elongation arrest (see above) nevertheless promotes translocation in the presence of an intact SRP receptor ( ). The ribosomes, however, do not appear to be bound to the SRP receptor (103). Instead, they become associated with two integral membrane glycoproteins, ribophorins I and II (56, 112), which in turn are probably associated with other membrane proteins. Ribophorins are characteristic glycoproteins of the rough endoplasmic reticulum which copurify with ribosomes and SRP receptor and can be cross-linked to membrane-bound ribosomes. Interaction of ribophorins I and II with each other and with cytoskeletal proteins may contribute to the maintenance of normal endoplasmic reticular organization, but specific functions for these proteins in transmembrane translocation have not yet been clearly defined. MICROBIOL. REV. C. Signal Sequence Receptor in the Endoplasmic Reticular Membrane Recently Wiedmann et al. (330) have provided evidence for the presence in the endoplasmic reticulum of a 35-kDa integral membrane receptor which binds the signal sequences of secretory proteins. In these studies a crosslinking approach was used which was analogous to that in which the 54-kDa protein of SRP was shown to function in signal recognition. A functional complex between the endoplasmic reticular membrane and the ribosome- and SRPbound arrested fragment of preprolactin was formed, preventing release from elongation arrest by cycloheximide. Subsequent exposure to irradiation yielded a cross-linked integral membrane protein product with a molecular mass of about 43 kda. Since the modified arrested fragment of preprolactin had a molecular mass of 8 kda, the membrane protein was presumed to have a molecular mass of 35 kda. The formation of this 43-kDa cross-linked product was prevented by prior treatment of the membranes with N- ethylmaleimide or a protease, but protease-treated membranes to which the cytoplasmic domain of the SRP receptor (a 60-kDa fragment) was added restored the appearance of the cross-linked product. Surprisingly, the cross-linked product was still capable of further polypeptide chain elongation if translation was continued after irradiation. Evidence was presented suggesting that the membrane protein which bound the signal sequence was a glycoprotein, and it was designated the signal sequence receptor (330). The results argue against an exclusive interaction of the signal peptide with the lipid constituents of the bilayer and suggest an involvement of at least two integral membrane proteins in addition to the ribophorins in the secretory process. Furthermore, studies analyzing translocation intermediates point toward an involvement of guanosine triphosphate-binding proteins in the segregation of proteins into the endoplasmic reticulum (54, 119). Whether these proteins, or the glycosylation site-binding protein (100), are members of the postulated channel (23) remains to be determined. III. INSERTION OF INTEGRAL MEMBRANE PROTEINS INTO THE ENDOPLASMIC RETICULUM The insertion of many integral proteins of the endoplasmic reticular membrane apparently involves an SRP-dependent mechanism (Table 1). This fact provides some of the best evidence that integration of membrane proteins into the phospholipid bilayer has a common mechanism with the process whereby proteins are secreted across the membrane. In one case, that of the small cytochrome b5 protein, a requirement for SRP could not be demonstrated (9). Small soluble proteins are secreted into the endoplasmic reticulum by an SRP-independent mechanism (see section II.A), and small bacterial proteins are integrated into the E. coli membranes by a SecA-SecY-independent mechanism (see section V.A), suggesting a functional analogy. Also revealed by the results summarized in Table 1 is the fact that in only one of the cases cited, that of the B-subunit of the acetylcholine receptor, is the signal sequence cleaved to yield a processed, mature protein. It is therefore clear that either noncleavable N-terminal signal sequences interact with the SRP or internal sequences serve as the SRP recognition sites (12, 93, 166). It should be noted that a direct interaction between the SRP and a signal sequence of one of the integral membrane proteins of the endoplasmic reticulum has not yet been demonstrated in chemical cross-linking studies (see section II.A). However, SRP-mediated translational arrest has been demonstrated for most of the proteins listed in Table 1. The nature of the signals responsible for insertion of polytopic (multiply transmembrane) integral membrane proteins into biological membranes has been considered by several workers (see, for example, reference 309 for a recent review). The signals reside in and around the transmembrane segments of integral membrane proteins and have been termed topogenic sequences (22). They are defined as discrete regions that allow initiation (signal sequences) and termination (stop transfer sequences) of transmembrane

5 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 337 TABLE 1. SRP-dependent membrane insertion of proteins Probable Integral membrane Probable no. of locationa of: Signal SRP- SRP-mediated protein transmembrane sequence dependent translational Reference(s) segments Amino Carboxy cleavage insertion arrest terminus terminus Acetylcholine receptor (8-subunit) In Out Ca2+-ATPase ca. 7 In Out ; J. Kyte, personal communication Lens membrane protein MP26 6 In In ; B. Gilula, personal communication Cytochrome P Sindbis virus PE2 Out In Coronavirus El ca. 3 Out In Opsin 7 Out In Asialo-glycoprotein receptor 1 In Out , 282 Glucose transporter 12 In In Invariant chain 1 In Out Transferrin receptor 1 In Out y-glutamyl transpeptidase 1 In Out Influenza A virus M2 1 Out In Cytochrome b5 1 In Out a In, Intracellular; Out, extracytoplasmic. polypeptide translocation, respectively. The topogenic function of membrane-spanning segments has been demonstrated by constructing fusion proteins between these sequences and reporter proteins. These fusion proteins were sometimes found to be translocated completely across membranes (27, 343), but translocation of other fusion proteins was interrupted (251, 341). A stop-transfer sequence always leads to anchorage of the polypeptide chain within the lipid bilayer, whereas a signal sequence does so only if it is not cleaved during assembly of the protein. Whether cleavage of a signal sequence occurs depends on structural constraints imposed by the flanking regions, which either render a potential cleavage site cryptic (167, 168) or influence the orientation of the signal sequence within the lipid bilayer (287). In general, the actual location of a given transmembrane segment within a polypeptide chain appears to influence its topogenic function. In some instances an internalized signal sequence mediates translocation of flanking regions (199, 268), whereas in other instances it does not do so unless the upstream sequences are short (91). Stop-transfer sequences usually halt translocation initiated by a preceding signal sequence. However, they may function as signal sequences if no other topogenic sequence occurs in the upstream region (177, 239, 342). A second internal signal sequence can function as a stoptransfer sequence (52), although this appears to be the exception (91, 239). The question then arises of which factors determine how the topogenic information is decoded. Mize et al. (177) have presented evidence that a stop-transfer sequence can interact with both SRP and an N-ethylmaleimide-sensitive receptor of the endoplasmic reticular membrane when it is placed in the position of an authentic signal sequence. It is therefore possible that stop-transfer information is decoded by appropriate membrane receptors, although an alternative possibility has been proposed (63, 82). According to Davis and Model (63), the thermodynamics of interactions between the hydrophobic amino acyl side chains of the translocated protein and the hydrophobic core of the phospholipid bilayer determine termination. If, however, topogenic sequences are recognized by membrane receptors, it is likely that the ensuing response depends on factors affecting the ligandreceptor interaction, such as the orientation of the transmembrane segment and steric constraints imposed by flanking regions (10). The occurrence of multiple topogenic sequences within polytopic, transmembrane proteins has led to the conclusion that their proper insertion results from the sequential decoding of signal and stop-transfer information encoded within the polypeptide chain. The final topology of the protein within the membrane is therefore dependent on the order as well as the nature of the topogenic sequences (239). It has been suggested that only the first of the individual translocation steps requires SRP (326). IV. TARGETING TO THE ENVELOPE FRACTION OF BACTERIA: CONSTITUENTS OF THE PROTEIN INSERTION-SECRETION SYSTEM As noted above for secretory proteins targeted to the endoplasmic reticulum of eucaryotic cells, bacterial proteins destined for either the periplasmic or outer membrane compartment of the E. coli envelope possess characteristic N-terminal signal sequences. Like their eucaryotic counterparts, these presequences are about 20 residues in length, consisting typically of a positively charged N terminus, a central hydrophobic region, and a flexible C-terminal region frequently predominating in hydrophilic residues (98, 304). The core hydrophobic region is frequently of about the length required to penetrate a bilayer as an a-helix. Despite these similarities, Gascuel and Danchin (98) have pointed out statistically significant differences between the procaryotic and eucaryotic presequences which may conceivably have mechanistic significance. For example, the hydrophobic cores in the eucaryotic signals are believed to be more rigid, but constraints at both ends of these core regions in the bacterial signals are more stringent. Despite these differences, the facts that the bacterial secretory machinery can accommodate eucaryotic proteins and even correctly process them and that the eucaryotic machinery can similarly translocate and process bacterial proteins (see section IV.C) argue in favor of conservation of function for the systems in the two major kingdoms. The recent demonstration (with appropriate controls) that a synthetic peptide corresponding

6 338 SAIER ET AL. to the signal sequence of the LamB maltoporin inhibited the translocation of precursors of periplasmic and outer membrane proteins into cytoplasmic membrane vesicles at a concentration of 1,uM argues for the autonomy of these sequences in receptor binding (47). The involvement of these leaders both in receptor recognition and in transmembrane translocation and the genetic dissection of these two functions from one another (293) demonstrate the functionally complex nature of these short sequences. The extent of our current knowledge regarding the protein constituents of the bacterial translocation machinery is evaluated in this section. The possible involvement of specific lipid constituents of the membrane (65, 80, 161) should not be forgotten. A. Probable Antifolding Receptor Proteins: the SecB Protein, Trigger Factor, and the Heat Shock GroEL Protein of E. coli The secb gene maps on the E. coli chromosome at 80.5 min near other genes encoding proteins involved in cell envelope biogenesis (gpsa and rfa). The initially isolated mutants defective for the SecB protein produced a low-level secretion defect, accumulating precursors of some exported proteins in the cytoplasm (148). Chromosomal secb::tns mutants which lack secb function altogether were subsequently shown to possess a nonlethal phenotype when grown on minimal media (149). In fact, the mutant bacteria grew nearly as well as the parental strain did in a minimal medium with glycerol as the sole source of carbon and energy. Thus, the SecB protein is nonessential under some growth conditions. Surprisingly, the mutation was reported to be lethal when the bacteria were grown on a complex medium (LB broth plates), but growth in liquid LB broth has been observed by other workers (G. A. Daniels and M. H. Saier, Jr., unpublished results). Examination of the specificity of the secretory defect revealed that the export of some precursor proteins, those giving rise to the periplasmic maltose-binding protein (MalE) and two outer membrane porins (OmpF and LamB), was defective, although accumulation of precursor forms of the periplasmic ribose-binding protein and alkaline phosphatase could not be demonstrated (149). Similarly, export of the major E. coli lipoprotein was not affected by the secb null mutation (319). It appears that null secb mutations exhibit differing degrees of secretory block depending on both the protein under study and the culture conditions used. In a recent report, Collier et al. (53) have provided evidence that the SecB protein facilitates export of the maltose-binding protein by virtue of an antifolding activity associated with the protein. Neither the naturation of newly synthesized proteins nor the unfolding of partially natured proteins is well understood (334). It has recently been demonstrated that the leader peptides of two periplasmic precursor proteins (the maltose and ribose precursor-binding proteins) can function to substantially retard the folding process (198). It is possible that the leaders influence folding by interacting with portions of the mature protein prior to acquisition of the native state (170, 220). The maltose-binding protein of E. coli is an example of a protein which has been shown to be capable of both co- and posttranslational modes of export (134, 242, 323). Randall and Hardy (219) showed that folding of previously synthesized maltose-binding protein precursor to a state resembling its native conformation, as measured by loss of protease sensitivity, was correlated with loss of export proficiency in vitro. The work of Collier et al. (53) and, more recently, of MICROBIOL. REV. Kumamoto and Gannon (151), Weiss et al. (323, 324), Kumamoto et al. (150), and Gannon et al. (95) has led to the suggestion that the SecB protein functions to maintain an export-competent state of the maltose-binding protein precursor by interacting with sites in the mature region of the protein and thereby retarding premature acquisition of its native conformation. A function of this nature was first suggested by the finding that synthesis of various exportdefective male mutant proteins interfered with normal protein export by limiting SecB availability. Synthesis of the export-defective malea323 gene product, deleted for the last 20 amino acyl residues of the signal sequence (including the hydrophobic core) and the first 89 residues of the mature protein (17), interfered with export and maturation (signal peptide cleavage) of the MalE and OmpA protein precursors in vivo as revealed by [35S]methionine pulse-labeling studies (53). Further experiments with a variety of mutant MalE proteins and various fusion proteins showed that synthesis of a protein which contained only the first 140 residues of the mature maltose-binding protein, with or without the presequence, did not interfere with normal protein export, but extension of the polypeptide chain to include the first 186 residues did cause interference. The results obtained with a variety of deletion mutants suggested that the region between residues 151 and 186 of the native maltose-binding protein was primarily responsible for the interference phenomenon. Of greatest importance was the finding that overproduction of the secb gene product, encoded on a multicopy plasmid, eliminated interference, suggesting a direct role of SecB in the process. As might be expected, only export of proteins shown previously to be secreted in a SecB-dependent fashion (for example, the OmpA and LamB proteins) was adversely affected in cells synthesizing an export-defective, SecB-consuming, maltosebinding protein. Other proteins, shown previously to be secreted normally in secb mutants (see above), were not subject to interference. These proteins may be secreted in a tightly coupled cotranslational process which is independent of SecB, or they may use an alternative receptor protein with overlapping function. The former proteins may be secreted by a loosely coupled, cotranslational process or a posttranslational process which allows cytoplasmic folding before completion of export (53, 151). The SecB protein presumably inhibits the naturation process, thereby prolonging the time allowed between translation of a polypeptide moiety and transmembrane translocation. The antifolding activity of SecB was inferred from the results of three distinct types of experiments. (i) The defect in maltose-binding protein precursor export in secb mutants was suppressed by mutational alterations in male which accelerated export, most probably because they reduced the rates of folding of the mutant proteins (53, 55). (ii) Export of a mutant MalE protein with an altered regional sequence that had been shown to cause export to occur in a strictly posttranslational manner (243) was totally dependent on SecB function (53). (iii) The rate of folding of the wild-type maltose-binding protein precursor, synthesized in vitro (monitored by measuring the rate of acquisition of proteinase K resistance [219]), was shown to be retarded by the presence of excess SecB protein but accelerated by the absence of SecB. Although these results have recently been confirmed and extended (150, 324), there is no evidence that SecB can induce unfolding of a protein once it has assumed a stable, protease-resistant conformation (53, 151, 324). These results, taken together, strongly suggest that export-defective maltose-binding protein precursor species

7 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 339 which tightly bind to the cytoplasmic SecB protein, but cannot readily dissociate from it because export is impeded, titrate out the SecB protein and render it the rate-limiting component for protein export. A pleiotropic defect in the export of a subset of proteins results. Interestingly, the SecB protein has been shown to associate with nascent chains of the pre-ribose-binding protein in vitro, even though export of this protein was not found to be SecB dependent in vivo (C. A. Kumamoto, personal communication). As noted above, binding of SecB to the precursor of the maltose-binding protein appears to occur outside the signal sequence. This fact was recently demonstrated for other export proteins as well. The export of the SecB-independent major lipoprotein could be rendered SecB sensitive by replacing the NH2 terminus of the lipoprotein precursor by the signal sequence plus 11 amino acids of mature OmpF. The resulting defect in the export of the hybrid protein observed in the secb null mutation, however, was quantitatively much less than that observed with the authentic OmpF protein, indicating that more of the mature OmpF than the N-terminal 11 amino acyl residues was required for efficient interaction with SecB (319). In addition, certain exportdefective signal sequence mutations of the outer membrane protein LamB can be suppressed by a distinct mutation in the secy gene locus (see section IV.D) suggesting a direct interaction between SecY and the signal sequences. Trun et al. (298) recently showed that this suppression was eliminated by the SecB null mutation, suggesting that both SecB and SecY interact with pre-lamb. Since signal sequence mutations that block interaction with wild-type SecY do not affect recognition by SecB, as visualized by the SecB mediation of translocation in a SecY suppressor strain, a direct interaction between SecB and the signal sequences is unlikely. These results therefore support the view that SecB recognizes sequences outside the signal sequence. They are in agreement with an earlier report (17) suggesting that a sequence within the maltose-binding protein plays a role (albeit possibly passive) in the secretory process. This role appears to involve a transient interaction with the SecB protein. Whether the primary function of the SecB protein is to stabilize an open or unfolded conformation of the precursor protein or whether this is merely a secondary effect of its binding to the precursor has yet to be determined. Nevertheless, the results serve to attribute the first clear biochemical function to a product of a genetically identified component of the secretory apparatus in E. coli. The recent purification of SecB to homogeneity (150, 324) and the demonstration that it consists of a multimeric protein composed of 17-kDa subunits with a native molecular mass of about 90 kda should allow more detailed biochemical studies of its function and definition of its interactions with other components of the secretion-insertion machinery. Crooke and Wickner (59) have identified a soluble protein, which they termed trigger factor, that interacts with in vitro-synthesized pro-ompa (precursor of OmpA) and appears to stabilize the translocation-competent form of this precursor. Just as the SecB protein interacts with a mature portion of the maltose-binding protein, trigger factor may interact with a mature part of the pro-ompa protein. When plasma membrane vesicles were used, pro-ompa, purified in the presence of 8 M urea, renatured and lost its translocation competence after removal of urea unless a soluble protein fraction containing trigger factor was added (59). The complex of trigger factor with pro-ompa was more sensitive to limited proteolysis than was the translocation-incompetent form of pro-ompa. Translocation required trigger factor, ATP, a transmembrane electrochemical potential, and SecY (57). Approximately 10% of the purified pro-ompa added was translocated. It is not clear that the action of trigger factor in preventing renaturation of pro-ompa in vitro adequately represents its function in vivo. Crooke et al. (58) purified a large amount of the pro-ompa protein and conjugated it to a Sepharose matrix to allow affinity isolation of trigger factor. The amino-terminal 12 amino acyl residues of the purified trigger factor were determined by Edman degradation, and the sequence was shown to be different from those of published Sec proteins and other proteins in the 1987 data base. The molecular mass was determined to be 63 kda. Trigger factor and pro-ompa were shown to form a 1:1 complex which could be isolated by gel filtration. The authors also reported that SRP from canine pancreas microsomes, like trigger factor, stabilized the translocation-competent conformation of pro-ompa (58). This conclusion was drawn from the finding that the detergent Nikkol prevented incorporation of the OmpA precursor into E. coli membrane vesicles when this protein was rapidly renatured following urea removal. This inhibitory effect of Nikkol could be overcome by SRP. Strictly reasoned, two different events were being compared: the preservation of translocation competence by trigger factor, and relief from detergent-mediated translocation incompetence by SRP. Moreover, restoration of only 2% of the translocation efficiency by SRP was observed and may not be significant. In any case, it will be interesting to find whether trigger factor and the SecB protein exhibit the same function with different protein specificities and whether SRP serves this function in eucaryotes. Trigger factor can be isolated from 70S ribosomes of E. coli (163). The LiCl-dissociated protein reassociates with the salt-washed 50S ribosomal subunit, which is known to contain the exit site for nascent polypeptides. Soluble trigger factor was also shown to compete with the pro-ompatrigger factor complex for a site in the membrane which is required for pro-ompa translocation (163). The results suggest a functional analogy with SRP (see section II.A) or eucaryotic cytoplasmic proteins (51, 64, 328) and also with the S complex of gram-positive bacteria (see section IV.F). Strengthening this postulate is the observation that either SRP or E. coli trigger factor plus yeast cytosol can support translocation of purified pro-ompa across yeast or canine pancreas microsomal membranes (253). A third antifolding protein of E. coli, the heat shock GroEL protein, has recently been identified (24). A photocross-linking approach was used to show that this cytosolic, 20S tetradecameric protein (subunit molecular mass of 65 kda) formed a complex with newly synthesized, unfolded pre-,b-lactamase or chloramphenicol acetyltransferase. Like SecB and trigger factor, which stabilize the unfolded forms of other proteins, the GroEL protein appeared to stabilize the unfolded conformations of pre-4-lactamase and chloramphenicol acetyltransferase. GroEL, which possesses adenosine triphosphatase (ATPase) activity, was examined for the effect of ATP on the stability of the GroEL-pre-p3-lactamase complex. ATP, but not its nonhydrolyzable analog adenylyl (B--y-methylene) diphosphonate (AMP-PCP), caused dissociation of the complex. When the isolated GroEL-pre-p-lactamase complex was added to membranes, the pre-p-lactamase translocated across the membrane in the presence of ATP but not in its absence or in the presence of AMP-PCP (24). Evidently the GroEL protein can hold appropriate secretory proteins in a

8 340 SAIER ET AL. translocation-competent form. It therefore appears that SecB, trigger factor, and GroEL serve as soluble receptor proteins that bind newly synthesized secretory and integral membrane proteins either post- or cotranslationally, transport them to the integral membrane translocation machinery on the inner surface of the cytoplasmic membrane, and then release them as transmembrane translocation is initiated. A similar converging pathway has been suggested for protein import into mitochondria (203) (see section VII.C). The GroEL heat shock protein, like the DnaK heat shock protein of E. coli (18), may resemble the family of 70-kDa, ATPbinding heat shock proteins of S. cerevisiae and other eucaryotes which have been implicated in the posttranslational transmembrane translocation of precursor polypeptides across both mitochondrial and endoplasmic reticular membranes (51, 64). The subunits of some cytoplasmic, oligomeric proteins apparently lack the inherent ability to correctly assemble into their native states. The correct, posttranslational assembly of these polypeptides is promoted by a ubiquitous class of conserved proteins termed chaperonins. The groel gene product has been shown to be one such chaperonin (117). Both this protein and the GroES protein are required for the morphogenesis of certain bacteriophages, for deoxyribonucleic acid (DNA) replication, and for the correct assembly of the Rhodospirillum rubrum enzyme ribulosebisphosphate carboxylase/oxygenase (Rubisco) in E. coli (106). The GroEL protein of E. coli exhibits structural similarities to and extensive amino acyl sequence homology with the so-called Rubisco-binding protein of eucaryotic chloroplasts, which is involved in the assembly of the oligomeric chloroplast Rubisco (241). It can therefore be concluded that the GroEL protein and, consequently, possibly other antifoldases such as SecB and trigger factor may function in capacities other than translocation across or insertion into biological membranes. This fact implies that these proteins should not be considered merely to be receptor components of the secretion-insertion machinery of the cell. B. The SecA Protein Temperature-sensitive seca mutants with a lethal phenotype at the nonpermissive temperature (42 C) have been isolated and characterized ( ). The gene, present on the E. coli chromosome at 2.5 min, encodes a soluble, peripheral membrane protein of about 100 kda, which is required for the secretion of almost all periplasmic and outer membrane proteins tested so far (15, 190, 285). Precursors of secretory protein accumulate in the cytoplasm of temperature-sensitive seca mutants at the nonpermissive temperature. Recent results indicate that SecA is required for the insertion of certaih integral proteins of the inner, cytoplasmic membrane as well (signal peptidase, the SecY protein, and the mannitol permease; see section V). It is not required for insertion of the small M13 or PF3 phage coat proteins (145, 146, 277; A. Kuhn, personal communication) or for export of the small prepromelittin in E. coli (51a). However, increasing the sizes of these proteins frequently renders their membrane insertion or their transmembrane secretion dependent on SecA and SecY (51a, 145, 146) (see section V.A). Mutations which abolish the activities of certain known or suspected components of the secretory apparatus (SecA, SecD, or SecY) or conditions which result in the blockage of generalized protein secretion (such as occurs following overproduction of certain male-lacz gene fusion products) result in substantial (>10-fold) overproduction of the SecA protein MICROBIOL. REV. (195, 231). This effect is apparently due exclusively to an increase in the rate of translation, since the levels of the seca mrna are not altered by these mutations and conditions (263). It appears that the SecA protein autogenously regulates its own translation by an unknown mechanism. seca is cotranscribed with a gene of unknown function, gene x, located upstream of seca as revealed by the fact that a nonsense mutation of gene x results in a SecA- phenotype (264). Secondary-site mutations which partially overcome the secretory defects in seca mutants with low residual SecA protein activity have been found to do so by decreasing the rate of protein synthesis (157, 191). It appears likely that decreased protein synthesis merely allows the residual activity of the defective SecA protein to function in the normal transport of essential exported proteins by preventing saturation and clogging of the export apparatus with a multitude of rapidly synthesized proteins. Recently the seca gene has been sequenced, and the deduced sequence of the encoded gene product has been analyzed (264). Sequence analysis of the SecA protein revealed that it consists of 901 amino acyl residues with a predicted molecular weight of 101,902. This latter value is close to the value of 92,000 determined biochemically (194). Hydropathy analyses indicated that the protein should be water soluble, consistent with its known peripheral association with membranes and ribosomes. The protein did not show significant homology with any other sequenced protein of bacterial, eucaryotic, or viral origin analyzed. Nine seca(ts) mutants, all defective for protein secretion, were sequenced to determine the sites and nature of the mutations. All of these mutations were single-point mutations tightly clustered at three sites within the first 170 amino acyl residues of SecA. The nature of these amino acyl substitutions was such that they were expected to alter protein secondary structure or cause a change in the hydrophobic-hydrophilic characteristic of a local region of the protein. Since mutations in only three regions in the first 20% of the seca gene were obtained, it is possible that the SecA protein is multifunctional and plays more than a single role in the secretion-insertion process. additional seca (prid) mutants In a more recent study, were isolated (90) that suppressed a number of male mutations, all of which had previously been shown to give rise to maltose-binding protein precursors with defective signal sequences. Three such suppressor mutations were shown to represent amino acyl substitutions at residues 111, 373, and 488 of the 901-residue SecA protein. The essentiality of the SecA protein for translocation has recently been demonstrated in vitro. Cabelli et al. (38) showed that removal of the SecA protein by passage of a cytoplasmic fraction from wild-type E. coli through an anti-seca antibody column abolished protein translocation of pro-ompa and the precursor of alkaline phosphatase into membrane vesicles derived from a seca amber mutant. Submicromolar quantities of the SecA protein restored activity. Translocation-defective in vitro systems containing urea-treated membranes, as well as similar SecA-dependent systems (E. Swidersky and M. Muller, manuscript in preparation), now allow a thorough biochemical investigation of SecA functions. SecA has been purified to apparent homogeneity from an overproducing strain by using either ATP-Sepharose or phosphocellulose for affinity chromatography (59a, 137). The protein was isolated in soluble form, but it binds to membranes with high affinity and supports translocation of the

9 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 341 pro-ompa protein in the presence of trigger factor. Interestingly, pro-ompa was found to bind to membrane vesicles either with or without the SecA protein, but only when SecA was bound to membranes prior to pro-ompa did translocation occur (59a). A most interesting property of SecA revealed by studies with the purified protein is that it hydrolyzes ATP (164). A universal ATP dependency of both co- and posttranslational transmembrane polypeptide translocation in both procaryotic and eucaryotic systems is well established (see references 45 and 46; Tai, in press; and section IV.C for a detailed consideration of the evidence). Use of an in vitro system with purified pro-ompa and SecA as well as urea-inactivated, inside-out E. coli vesicles showed that ATP hydrolysis activity was dependent on all of these components. In the absence of any one of these components, the inherent ATPase activity of the SecA protein was reduced to a minimal value. The stimulatory activity of the membrane vesicles could be blocked by using antibody to the SecY protein or by preparing the membranes from a thermosensitive secy mutant which had been incubated at the nonpermissive temperature in vivo. The SecA protein might possess more than one ATP-binding site, since 8-azido-ATP inactivated SecA for pro-ompa translocation and translocation-dependent ATPase activity but did not inhibit the low basal ATPase activity inherent to the protein (164). The results suggest that SecA plays a central role in coupling ATP hydrolysis to transmembrane polypeptide translocation. Quantitative determination of the stoichiometry of ATP hydrolysis with translocation under optimal conditions may reveal important mechanistic aspects of the translocation process. C. Soluble Export Factors from E. coli In an early study, Muller et al. (184) demonstrated that the in vitro transcription of E. coli pbr322 DNA could be coupled to cell-free translation in a wheat germ system. Pre-1-lactamase, encoded on pbr322, was the major translation product. Addition of canine pancreas microsomes resulted in successful translocation into the microsomes and processing to mature 1-lactamase, as shown by N-terminal sequence analysis. Processing was dependent on microsomal vesicles as well as SRP, which also caused partial inhibition of translation in this heterologous procaryoticeucaryotic transcription-translation-translocation system. The results clearly indicate that secretion and processing of procaryotic precursor proteins require the same functional protein constituents as for the endoplasmic reticulum-directed transport apparatus of mammalian cells. The apparent success of the eucaryotic in vitro cotranslational translocation experiments described above led Muller and Blobel (181) to develop an analogous, strictly bacterial system. Previously developed, less well-defined bacterial in vitro systems are discussed by these investigators (181). When inverted membrane vesicles from E. coli and three plasmid-encoded periplasmic or outer membrane precursor proteins were used, conversion to the mature forms could be demonstrated. Moreover, maturation was accompanied by association of the proteins with the vesicles in a proteinase K-resistant state owing to entry into the intravesicular space. Translocation in this in vitro system occurs both coand posttranslationally (181). The initial low efficiency of translocation has recently been significantly increased by varying conditions so that close to 50% of added LamB protein is routinely translocated (E. Swidersky and M. Muller, unpublished data). This increase in efficiency, however, applies only to the cotranslational mode of translocation. The versatility of the in vitro system was convincingly demonstrated by its applicability to the complex processing events to which the Braun lipoprotein is subjected (144, 294). This lipoprotein is initially synthesized as a 78-amino-acid precursor, which, upon transport across the plasma membrane, is modified by the attachment of a diglyceride moiety to the amino acid adjacent to the signal sequence cleavage site. This modification is a prerequisite for recognition by the unique signal peptidase II, which can specifically be inhibited by the antibiotic globomycin (335). Krishnabhakdi and Muller demonstrated that the lipoprotein precursor, when synthesized in vitro, was converted in the presence of inverted membrane vesicles to the mature form and that this event was abolished by globomycin (144). Maturation therefore must have been mediated by signal peptidase II. This fact, in turn, presumably reflects a previous lipid modification reaction occurring in this in vitro system. Further evidence for the relevance to the in vivo situation of the export events occurring in the E. coli in vitro system was provided by integration of the lactose permease, a polytopic transmembrane protein, into the lipid bilayer of the membrane vesicles. A functional permease resulted (4a; see section V.C). A similar cell-free export system was developed independently by Tai and coworkers (48, 223), and these bacterial synthesis-translocation systems have been used successfully by several groups (66, 101, 222, 340). Running the in vitro system in a posttranslational manner, Chen and Tai (44) were the first to demonstrate the need for hydrolyzable ATP in bacterial protein export. The additional requirement for a second source of energy, i.e., the proton motive force, was subsequently shown for the in vitro membrane transport of a multitude of proteins (66, 101, 183, 338). Although the role of ATP is now well established, the nature and specificity of the proton electrochemical gradient dependency are still controversial (4a; Tai, in press). In E. coli it seems to be required for the translocation of some proteins but not others (156; Tai, in press). It should be pointed out that there are several possible explanations for the inability to demonstrate a proton motive force requirement in vitro, and the earlier in vivo evidence should therefore be recalled (16, 60-62, 81, 347). The initial studies in which a heterologous procaryoticeucaryotic in vitro system was used prompted attempts to further interchange components from either source. E. coli plasma membrane vesicles and mammalian microsomes do not function with soluble lysates from the heterologous system for the secretion of procaryotic and eucaryotic secretory proteins (128; M. Muller and G. Blobel, unpublished data). However, Fecycz and Blobel (89) have shown that the bacterial in vitro system translocates and correctly processes yeast prepro-ot-factor. The soluble and membrane fractions from S. cerevisiae and E. coli were functionally interchangeable. Furthermore, Lecker et al. (156) have shown that secretion of prepro-a-factor occurs in a process which requires the membrane potential and the SecY protein, and some of the topological requirements were defined. Moreover, the human erythrocyte glucose transporter has recently been expressed in vivo in E. coli in a topologically and functionally correct fashion (254). These observations allow the conclusion that the procaryotic and eucaryotic systems must resemble one another from a functional standpoint. However, they also demonstrate specificity in the

10 342 SAIER ET AL. requirements for components of the respective translocation machinery. A reconstituted version of the in vitro system of Muller and Blobel (181) led to the discovery of a soluble factor, not present in or on the vesicles, which was required for transmembrane translocation. Extensive subfractionation of the soluble part of the E. coli in vitro synthesis-translocation system and subsequent reconstitution of protein synthesis from largely purified components obtained therefrom resulted in a significant reduction in the translocation of newly synthesized proteins into membrane vesicles. Partial purification and characterization of this soluble export factor revealed that it exhibited a sedimentation coefficient of about 12S and showed tendencies to dissociate (182). This last property, together with its recently observed translational arrest activity (B. Drees and M. Muller, unpublished data), has made its purification to homogeneity a difficult task. No small cytoplasmic RNA (see section IV.G) has so far been found to be associated with active preparations of the factor. Recently Watanabe and Blobel (318) constructed a mutant form of the maltose-binding protein precursor which, unlike its wild-type counterpart, does not undergo naturation to an import-incompetent form. In a homologous E. coli translation-translocation system, the mutant precursor was translocated both co- and posttranslationally with nearly 100% efficiency into inverted vesicles. By contrast, translation of the mutant precursor protein by a wheat germ system and subsequent exposure to inverted vesicles did not result in translocation. Addition of an E. coli postribosomal supernatant to this heterologous system greatly stimulated translocation of the mutant precursor. Although it appeared somewhat smaller (7S), the activity appeared to be due to the soluble export factor found by Muller and Blobel (182). It can bind the mutant maltose-binding protein precursor posttranslationally in the absence of ATP, although subsequent targeting to vesicles and translocation clearly required ATP. Purification was reported to reveal the presence of the SecB gene product (318). It was suggested that this factor functions as the E. coli equivalent of the eucaryotic SRP. Recently Weng et al. (325) used an ATP-dependent posttranslational protein translocation system to examine the soluble protein factors which might be involved. The translocation assay used was similar to the one developed for the identification of trigger factor (see section IV.A), but it differed from the latter assay in that it used an OmpA precursor which was only partially purified. It was inactive with the alkaline phosphatase precursor protein. When this in vitro system was used, two soluble factors which stimulated protein translocation were identified. Both were proteinase-k sensitive, micrococcal nuclease insensitive, N-ethylmaleimide insensitive, and heat labile. The major factor sedimented during sucrose density gradient centrifugation with a sedimentation coefficient of about 8S and exhibited a molecular mass of about 120 kda by gel filtration. The minor factor sedimented with a sedimentation coefficient of about 4S and had an apparent molecular mass of about 60 kda. Each factor stimulated the translocation of pro-ompa in a concentration-dependent manner, and the effects of the two in combination were roughly additive. To what degree these factors overlap with the factor described by Muller and Blobel (182) and Watanabe and Blobel (318) is currently not known. The availability of functional E. coli in vitro translocation systems offers the possibility of assaying and purifying the MICROBIOL. REV. soluble and membranous components of such systems by using export-defective mutant strains. Restoration of translocation in these mutant systems should be feasible by complementation with fractions from wild-type E. coli. In only one case (59a) has such a mutant system been used for purification of the proteins mediating translocation, and recently an in vitro system prepared from a seca(ts) mutant was exploited for the purification of a 15-kDa polypeptide constituent of the secretory system which is not related to SecA or SecB (B. Thome, H. K. Hoffschulte, S. Krishnabhakdi, and M. Muller, manuscript in preparation). This ribosome-associated protein enhanced the low level of translocation observed in a seca mutant extract. Further functional and structural analyses are required to gain an understanding of the physiological role of this protein, as well as its relationship to other E. coli translocation factors. D. The SecY Protein The secy locus was initially defined by the isolation of mutants which reversed the effects of secretory defects in strains containing lamb signal sequence mutations (78, 79). The mutations mapped to three unlinked loci, one of which (pria or secy) was located at 72 min on the E. coli chromosome, within the spc operon encoding ribosomal proteins and downstream from the last known ribosomal protein gene. Additional alleles of the secy gene were isolated following localized mutagenesis of this region of the chromosomal DNA and selection for mutants with temperaturesensitive secretory defects (131, 271). The temperaturesensitive sec Y mutants accumulated cytoplasmic precursors of several exported proteins following a shift from the permissive to the nonpermissive temperature. Extensive evidence was presented leading to the conclusion that secy and prla are allelic. Other mutations in the same gene were capable of partially suppressing defects caused by mutations in the seca gene (34). The secy gene has been sequenced (42) and encodes a hydrophobic protein of 443 amino acyl residues. The protein has an apparent molecular weight on sodium dodecyl sulfatepolyacrylamide gels that is lower than predicted on the basis of the amino acyl sequence, as expected for an integral membrane protein (5, 130). Hydropathy analysis of the deduced amino acyl sequence suggests a structure consisting of 10 transmembrane segments with 9 interspersed hydrophilic segments and with hydrophilic, cytoplasmically located segments at the N and C termini (5). These topological predictions are supported both by studies concerned with the susceptibility of the protein to protease digestion and by studies in which the orientation and localization of alkaline phosphatase in the cytoplasmic membrane were determined by using a collection of SecY-PhoA fusion proteins (5). Genetic data on sec Y mutants, especially those originally isolated as suppressors of signal sequence mutations, have been interpreted in terms of a direct interaction between SecY and the signal sequences. However, Randall et al. have pointed out that there are weaknesses in the genetic evidence (221). If a mutation in one protein can be overcome by a compensatory mutation in a second protein with which the first protein interacts (as has been assumed for suppressors of signal sequence mutations), one would expect that the resulting suppressor mutations would show allele specificity. With only two exceptions, all of the alleles of prla show the same predictable effect with several signal sequence mutations. Some of the mutant alleles were isolated as suppressors of mutants lacking all or most of the hydrophobic residues of the signal sequences. Furthermore, none of the alleles impair the export of proteins having wild-type

11 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 343 signal sequences. These properties led Randall et al. (221) to suggest that the mutations may suppress secretory defects in an indirect manner rather than by directly interacting with the signal sequences. Evaluation of the effects of mutations in secy should ultimately clarify the significance of particular amino acyl residues in SecY to its function. The locations of the amino acyl residues altered in several mutants has been determined. secy24, causing the accumulation of precursors of a number of periplasmic and outer membrane proteins, has Gly-240 changed to Asp (271). The mutation has been localized to a periplasmic, extramembrane loop of the protein (5). The wild-type, plasmid-encoded SecY protein can complement the protein translocation defect in cells containing the chromosomal temperature-sensitive sec Y24 mutation, indicating that the defect is limited to the SecY activity alone and does not impair other components of the machinery (13, 88). The reduction in translocation efficiency of secy24 strains can be mimicked by in vitro treatment of isolated vesicles at 40 C, suggesting that the mutant protein itself is heat labile (88). Evidence of compensation for the translocation defect in secy24 strains afforded by the addition of soluble factors has been obtained (88). The active constituent has recently been identified as the SecA protein (87), suggesting that SecY and SecA do, in fact, interact, thereby influencing the conformations and consequently the activities of each other. An interaction between SecY and SecB has also been proposed (298; see section IV.A). In a recent study by Sako and lino (252), additional pria (secy) suppressor alleles were cloned and sequenced. The four alleles identified had Phe-67 changed to Cys (prla3), Ser-282 changed to Arg (prla401), Phe-286 changed to Tyr (prla4-1), and Ile-408 changed to Asn (PrIA4-2). The prla4-1 and prla4-2 mutations were identified within the original prla4 mutant (283). In contrast to the previously known suppressor phenotype of pria mutations, the two mutations altering residues 282 and 286 (both in transmembrane segment 7) specifically blocked processing in E. coli of an exported enzyme from S. aureus, staphylokinase. By contrast, the C-terminal mutation which altered residue 408 did not block staphylokinase export but suppressed mutations in the pre-male and pre-lamb signal sequences. These results therefore strongly imply that the SecY protein does, in fact, interact directly with the signal sequences of exported proteins and that different regions of the SecY protein recognize different structural features of these sequences. Thus, transmembrane segment 7 may well interact with the staphylokinase signal sequence. It is interesting that secy mutations which suppress export defects resulting from alterations in the hydrophobic cores of signal sequences do not suppress defects resulting from alterations in the hydrophilic N- terminal segments of the same leaders (217). The phenotypes of secy mutants clearly indicate that the protein is important in secretion, but there are few indications about the function(s) of the wild-type protein. Because this is the only component among the secretory apparatus thus far identified that is an integral membrane protein, it is possible that the SecY protein functions as a proteinaceous pore through which the unfolded polypeptide is translocated (5). Evidence for such a possibility has been presented (21). It is interesting that SecY shows extensive sequence identity with an essential secretory protein of the yeast endoplasmic reticular system (Sec 61p) (C. Stirling and R. Schekman, personal communication). The E. coli SecY and the S. cerevisiae Sec 61p proteins, which are of the same length, exhibit 24% sequence identity between residues 70 and 200 in both proteins. Both proteins are polytopic integral membrane proteins with the same predicted pl values. However, the function of Sec 61p is not known, and the large number of integral membrane proteins involved in endoplasmic reticular secretion in eucaryotic cells reveals the multiplicity of functions possible for SecY. E. Other Possible Components of the E. coli Export System Defined by Genetic Analyses In addition to the constituents described above, two genetic loci (secd and sece) have been identified which appear to encode essential components of the secretory apparatus rather than ribosomal constituents, which only indirectly influence secretion (157). An additional locus (pric) encodes a nonessential protein, which may make up part of an alternative pathway for secretion (297). The secd mutations were shown to be cold sensitive for growth and to lead to accumulation of cytoplasmic precursors of exportable proteins at the nonpermissive temperature (97). The secd gene was shown to be closely linked to tsx at about 9 min on the E. coli chromosome. In agreement with the possibility that SecD is a component of the export machinery, secd mutations were shown to be recessive. The cold sensitivity of these mutants suggests that SecD may be an integral membrane protein, stabilized primarily by hydrophobic forces. Riggs et al. (226) have recently described additional mutants, one of which, like secd, exhibited a cold sensitive growth defect and a pleiotropic defect in protein export. The gene encoding this mutant, which maps at about 90 min on the E. coli chromosome, was designated sece. The presumed cold sensitivity of the SecE protein suggests that it may be stabilized largely by entropic forces and therefore may be an integral constituent of the membrane. Cloning and sequencing of the secd and sece genes may provide clues to their subcellular localization, but biochemical studies will undoubtedly be required to define their specific roles in the translocation process. The pric gene, mentioned above, maps at 71 min on the E. coli chromosome. Several different mutations in this gene have been shown to suppress certain signal sequence defects with alterations in the hydrophobic core (296). The strongest of these suppressor mutations, prlc8, restores processing of a mutant signal sequence to resemble that observed in the wild-type strain. Results obtained with various double mutants indicated that the PrlC protein can translocate the N termini of certain secretory proteins across the membrane in the absence of SecA, SecB, or SecY function, so that they can be processed by leader peptidase. The PrlC protein cannot, however, translocate the entire secretory protein across the membrane (297). How this protein participates in protein translocation under normal conditions is not known. F. Comparison of the Insertion Machinery of Gram-Positive Bacteria with That of E. coli Gram-positive bacteria differ from gram-negative bacteria in possessing a one-membrane envelope rather than the two-membrane envelope typical of E. coli. Frequently, these two classes of bacteria exhibit fundamental biochemical differences. For example, the components of the fatty acid synthase of E. coli are recoverable from broken-cell preparations as several distinct, easily separable, soluble or membrane-associated enzymes, whereas the same complex in the

12 344 SAIER ET AL. gram-positive mycobacteria is released from disrupted cells as a large, multifunctional, multienzyme complex (245). In this respect, the gram-positive synthase more closely resembles those from eucaryotes than those from gram-negative bacteria. In B. subtilis a 64-kDa protein associated with membranebound ribosomes was found that became accessible to antibody binding and proteolytic digestion only after removal of the ribosomes from the membranes (122, 123, 255). Anti-64-kDa protein antibodies precipitated a complex that included three additional proteins, of 60, 41, and 36 kda (41). This complex was termed the S (for secretion) complex because of its potential resemblance to the eucaryotic SRP. Its multimeric structure, the localization of the 64-kDa protein at ribosome-membrane contact sites, and the subsequent finding that it partitions between the soluble fraction and the membrane fraction all suggest an analogy with SRP. All of these characteristics except the multimeric nature of the complex also suggest an analogy with the SecA protein of E. coli. Strikingly similar findings were reported for S. aureus, which contains a 60-kDa protein of the same intracellular distribution as the 64-kDa B. subtilis protein. These two proteins exhibited antibody cross-reactivity. The 60-kDa staphylococcal protein was complexed with 71-, 46-, and 41-kDa proteins (1, 2). The intracellular level of this complex increased during the postexponential growth phase when the rate of protein secretion increased (3). The distribution of the complex between the cytoplasm and the membrane varied depending on the rate of exoprotein production, substantiating the suggestion that this complex functions in protein secretion. In a recent study (4) the genes encoding the four proteins of the S. aureus complex were cloned in E. coli and shown to be expressed from an 8.5-kilobase DNA fragment as judged by Western immunoblot analyses. The order of the genes on the cloned DNA fragment was determined. These four genes encoded (from left to right) the 46-, 41-, 71-, and 60-kDa proteins. A major, large mrna of the expected size, approximately 5.9 kilobases, presumably encoding all four protein components of the complex, was shown to be transcribed from it (4). Two minor and smaller mrna species (2.8 and 1.6 kilobases) were also shown to be transcribed from the cloned DNA fragment in the same direction (left to right). Sequence analyses of this cloned DNA should prove most interesting, as they may reveal homology with some of the eucaryotic and gram-negative bacterial constituents of the secretion-insertion systems. G. Bacterial RNA Species Showing Homology with Eucaryotic SRP RNA As discussed in section II.A, the SRP of eucaryotes contains a 300-nucleotide RNA molecule which links the protein constituents of the complex and may fulfill other functions as well. Recently, bacterial RNAs have been identified which show homology with SRP RNA (211, 224, 286). The one from the archaebacterium Halobacterium halobium resembles eucaryotic SRP RNA in every essential respect. E. coli and B. subtilis RNA species have been identified which also exhibit unique and highly conserved structural domains. These domains (analogous to domain IV of SRP RNA) are found in the so-called small cytoplasmic RNA of B. subtilis, with a size similar to that of SRP RNA, and in a 4.5S RNA species of E. coli, with a size about half that of the SRP RNA. The strong homology of these RNA MICROBIOL. REV. species suggests, first, a common evolutionary origin and, second, common functionality. Although the functions of the RNA species from H. halobium and B. subtilis have not been investigated, that from E. coli has been implicated in the process of translation and may interact with ribosomes and elongation factor G. It is essential for viability, and its abundance relative to that of the ribosome in E. coli is similar to that of SRP RNA in eucaryotic cells (211). These properties suggest that it has a possible role in the coupling of translation to polypeptide secretion-membrane insertion. It may be required only for the biogenesis of proteins which are obligatorily translocated across the membrane by a cotranslational mechanism in vivo. Further experiments are required to test these possibilities. V. INSERTION OF INTEGRAL MEMBRANE PROTEINS INTO THE CYTOPLASMIC MEMBRANE OF E. COLI Integral proteins of the outer membrane of E. coli, like periplasmic proteins, are initially synthesized with cleavable signal sequences resembling those targeting proteins to the endoplasmic reticulum of the eucaryotic cell. By contrast, with the exception of the M13 phage coat protein, integral proteins of the cytoplasmic membrane of bacteria, like integral membrane proteins of eucaryotes, are made without cleavable signal sequences, and N-terminal sequences which structurally resemble those of secretory proteins are usually lacking. Examples of well-characterized bacterial proteins with noncleavable N termini include the lactose permease (72), the MalF component of the maltose permease (94), the mannitol permease (159; B. Rippon, personal communication), the integral membrane subunits (Fo components) of the Fo-F1 ATPase (37), the leader peptidase (333), the SecY protein (5, 6), and the photosynthetic pigment proteins of phototrophic bacteria (68). Of these proteins, only the leader peptidase, the SecY protein, and the mannitol permease, have been investigated in detail with respect to the mechanisms of their insertion. Insertion of all of these proteins has been found to be dependent on the integrity of SecA and/or SecY, but not SecB (6, 331; G. A. Daniels, Y. Yamada, M. Yamada, and M. H. Saier, Jr., unpublished results). Insertion of at least some of these proteins is dependent on the membrane potential (332), although others are inserted in a potential-independent process (4a). Although the first of these three proteins possesses a typical (albeit short) noncleavable signal sequence characteristic of endoplasmic reticulum-targeted proteins of eucaryotes, the second possesses no characteristic presequence and the third possesses an amphipathic leader sequence similar to those of mitochondrion-targeted proteins of the eucaryotic cell (see sections VII.A and VIII.A). In a recent study, Baker et al. (15) examined the rates of incorporation of newly synthesized bulk protein into both inner and outer membranes of wild-type E. coli and two temperature-sensitive seca mutants of E. coli. In these experiments, cells were pulse-labeled with [35S]methionine at both the restrictive and permissive temperatures. At 300C all strains showed constant rates of protein incorporation into both inner and outer membranes. In the wild-type cells, the temperature shift to 42 C was without effect. In contrast, in the seca mutants the temperature shift resulted in immediate inhibition of protein incorporation into the two membranes. Incorporation of proteins into the two membranes was equally strongly inhibited, with residual activities being reduced to 30% of the control rates (15). These results suggest that in general, protein insertion into both the inner

13 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 345 and outer membranes of E. coli is dependent on SecA function. A. Insertion of Small versus Large Membrane Proteins As mentioned in section IV.B, small proteins are inserted into or secreted across the E. coli cytoplasmic membrane by a SecA-, SecY-independent mechanism (51a, 145, 146, 277; Kuhn, personal communication). Among these proteins are the two integral membrane coat proteins from phages M13 and PF3 and the water-soluble secretory protein prepromelittin, which is processed to the bee toxin, melittin. Kuhn et al. (147) showed that recombinant forms of the M13 procoat with either a leader sequence derived from the pro-ompa protein or a large carboxy-terminal extension retained their independence of SecY. The SecY independence of the recombinant protein in which the leader sequence of the procoat protein was exchanged for that of the pro-ompa protein revealed that SecY independence was not conferred by the amino acyl sequence of the N-terminal signal peptide. This conclusion follows from the experiment performed, since proper secretion of pro-ompa is SecY dependent. The fusion protein with a C-terminal extension (mentioned in the previous paragraph) had all but one of the residues of the procoat protein fused to a 103-amino-acyl polypeptide derived from the polar, C-terminal domain of the E. coli leader peptidase. The resultant fusion protein was correctly inserted into the membrane with its amino terminus facing the periplasm and its carboxy terminus remaining in the cytoplasm. The rate of insertion, however, was substantially lower than that for the normal M13 procoat protein (145, 147). In further studies (146), the extracellular domain of the M13 procoat protein was extended by construction of inframe insertions in which fragments of the ompa gene ranging from 294 to 522 base pairs were ligated with the procoat gene. Once again, the resultant hybrid proteins were inserted into the membrane and processed normally, but in this case the SecA and SecY proteins were required. From these experiments it can be concluded tentatively that a dependency of the insertion process on the seca and secy gene products is not determined strictly by the size of the protein being inserted, although the size and nature of the portion of the polypeptide chain which is translocated across the membrane may be a determining factor. The SecA-SecY machinery is apparently required for translocation of long and/or complex regions of a polypeptide chain across the membrane, but not for short, simple structures. It is interesting that the M13 procoat protein is processed by canine pancreas microsomes in vitro by an ATP-dependent mechanism in the absence of SRP and the docking protein (328). Two protein components, one of which is a 70-kDa heat shock protein, are required (346). The results suggest that at least in animal cells, and consequently possibly in bacteria as well, a unique assembly pathway for procoat protein and other small proteins exists. In animal cells this pathway does not appear to depend on the SRPdocking protein complex or the ribosome-ribophorin complex, but it does depend on ATP and two other proteins. In bacteria this postulated pathway would be independent of both SecA and SecY (328, 346). Results comparable to those reported above have been obtained with the secretory protein honeybee prepromelittin. This protein can be correctly processed and imported into canine pancreas microsomes (179, 345). The signal sequence of prepromelittin can interact with the SRPdocking protein system of the endoplasmic reticulum, but this interaction is not a prerequisite for secretion. Once again, the small size of this protein, and possibly its charge distribution as well, may be responsible for SRP independence. The SRP-independent secretory process was preceded by the initial binding of the precursor to the microsomal membrane surface and was ATP dependent (180). Cobet et al. (51a) have recently extended studies with this protein to E. coli. Prepromelittin secretion in E. coli was found to be both SecA and SecY independent. However, extension of this 70-residue precursor to 257 residues by fusion of its C-terminal moiety with mouse dihydrofolate reductase resulted in the synthesis of a polypeptide chain which was secreted by a SecA-SecY-dependent mechanism. The results seem to agree with those obtained with the M13 procoat protein, suggesting that a need for the known components of the secretory apparatus in both procaryotic and eucaryotic cells can be obviated when the secreted or integral membrane protein is sufficiently small. B. Charge Distribution as a Determinant of Integral Membrane Protein Topology As first suggested by von Heijne (305), the sign of the N-terminal charge of a topogenic sequence (either an amphipathic signal sequence of a mitochondrion-targeted protein; a hydrophobic signal sequence of a bacterial or an endoplasmic reticulum-targeted, secreted protein; or a transmembrane anchor sequence in an integral membrane protein which spans the membrane only once) may influence the orientation of the segment in the membrane (217, 309, 340). The same may be true of internal signal and anchor sequences in integral proteins which span the membrane several times (308, 310, 312). Thus, the frequency of positive charges associated with loops on the cytoplasmic membrane surfaces of these proteins greatly exceeds that of positive charges associated with loops on their external surfaces. This unequal distribution of positive charges associated with loops connecting transmembrane segments in a polytopic integral membrane protein correlates with the presence (inner loops) or relative absence (outer loops) of arginyl and lysyl residues within these structures. This generalization appears to apply not only to the proteins of the inner membrane of gram-negative bacteria, where basic residues occur four times more frequently in cytoplasmic loops than in periplasmic loops (308), but also to eucaryotic proteins of the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane (310). A "positive-inside" rule thus seems to apply to integral membrane proteins originating in all major cell classifications and organelles. The apolar, transmembrane segments apparently signify membrane integration, whereas the positively charged residues at the loop ends of these segments provide topological information, designating the orientation (sidedness) of the transmembrane segment in the bilayer. It can be presumed that the universality of this positive-inside rule reflects universal recognition features of the insertion machinery, although the possibility cannot be ruled out that an interaction with the membrane potential or with the asymmetric surface charge of a particular membrane plays a role. Evaluation of the C-terminal cleavage regions following the hydrophobic cores of N-terminal signal sequences of more than 200 procaryotic and eucaryotic exported proteins revealed a markedly higher incidence of acidic over basic

14 346 SAIER ET AL. residues in these regions (306). On the basis of this observation, it was suggested that positively charged segments adjacent to signal sequences are preferentially retained on the cytoplasmic side of the membrane, whereas negatively charged segments (or segments deficient in positive charge) adjacent to topogenic sequences are preferentially translocated across the membrane. Such an occurrence frequently correlates with preferential translocation in response to a membrane potential, but it may instead reflect the specificity of the insertion machinery, as noted above for the internal transmembrane segments of integral membrane proteins. This conclusion has recently been experimentally substantiated (162). In that study, positive charges were introduced in the N terminus of the mature portion of alkaline phosphatase, just following the hydrophobic core and cleavage site of the signal sequence. In one case, the presence of the sequence Arg-Ile-Arg in this region resulted in a 50-fold decrease in the extent of alkaline phosphatase export. Reduction of the net charge in this region, either by removal of positively charged residues or by introduction of negatively charged residues, resulted in enhanced export (162). In a recent study by Duplay and Hofnung (70), mutations in both the N terminus and the mature portions of the periplasmic maltose-binding protein of E. coli were shown to prevent release of these proteins from the membrane following translocation of at least their N-terminal moieties through the cytoplasmic membrane. These mutant proteins were not released from the bacterial cell by osmotic shock, and their overproduction was lethal, even though their N-terminal signal sequences were correctly processed by the signal peptidase. It was suggested that the two mutationally defined regions of the mature maltose-binding protein (residues 18 to 42 and 208 to 306) may be important to a conformational alteration in the translocated polypeptide which allows release from the cytoplasmic membrane (70). The idea that a conformational change in the mature protein is necessary for release had been put forth previously (142, 176). The results suggest, first, that release of a secreted protein from the cytoplasmic membrane may be more complex than previously thought and, second, that the retention of a normally soluble protein within the cytoplasmic membrane as an integral constituent may result from rather minor changes in the amino acyl sequence. These observations, or the charge effects noted originally by von Heijne (305, 306, 309) and more recently by Li et al. (162), may explain earlier results showing that P-galactosidase and several other soluble proteins could not be efficiently translocated across the E. coli cytoplasmic membrane, even when provided with signal sequences from naturally exported proteins. The results are in agreement with those reached by Andrews et al. (10) and Rothman et al. (239), as discussed in section III. They are likely to be explained by a single mechanism which follows the rules governing both the process of protein secretion across membranes and that of protein insertion into them in many if not all biological cells and organelles. C. In Vitro Membrane Insertion of the E. coli Lactose Permease Recently, insertion of the lactose permease was analyzed in vitro (4a). The protein was synthesized in an E. coli cell-free transcription-translation system and was found to become membrane associated upon addition of inside-out membrane vesicles to the synthesizing extract. Most strikingly, the vesicles thereby acquired the capability to accumulate lactose. This result demonstrated for the first time that a polytopic integral plasma membrane protein can assemble correctly in vitro into membrane vesicles to give rise to an enzymatically active transmembrane protein. Surprisingly, integration was independent of the proton motive force, which is consistent with the notion that only bacterial proteins whose export involves translocation of fairly long polar stretches across the plasma membrane require energy from a transmembrane ion gradient. VI. MICROBIOL. REV. TEMPORAL MODE OF PROTEIN TRANSPORT ACROSS CELLULAR MEMBRANES The debate about whether proteins cross membranes during (cotranslationally) or after (posttranslationally) their synthesis has been considered in detail by others (158, 171, 204, 323). Fundamental mechanistic differences between cotranslational and posttranslational pathways have not become apparent (278, 279). The same enzymatic machinery appears to be operative in both cases, both in bacteria (13, 158, 242) and in eucaryotes (278, 279). However, as noted above, the lactose permease, an inner membrane protein, when synthesized in vitro, integrated efficiently into membrane vesicles only cotranslationally (4a). Posttranslational integration also occurred, but to an extremely small extent, probably owing to aggregation of the hydrophobic enzyme in the membrane-free environment. In principle, there have been three experimental approaches to the problem of co- versus posttranslational transport. (i) Intact cells have been pulse-chase labeled, and the fate of a particular secretory protein has been monitored by immunoprecipitation. If the precursor form of the protein becomes visible during the pulse period, posttranslational processing is suggested. That this processing reflects translocation across the membrane is generally accepted. (ii) Coor posttranslational supplementation of an in vitro synthesis system with membranes allows one to approach the question of how much time can elapse before a precursor protein becomes transport incompetent. (iii) By screening for nascent polypeptide chains, one can determine the length of the growing chain which is required before the presequence can be cleaved (218). From these experimental approaches, it had become established that proteins generally enter the endoplasmic reticulum cotranslationally, that they enter mitochondria posttranslationally, and that both modes apply to the export of bacterial proteins. In full agreement with the concept of cotranslational transport, eucaryotic SRP, functioning in endoplasmic reticular targeting, can induce the phenomenon of translational arrest, achieving temporal coordination between synthesis and endoplasmic reticulum-targeted transport (see section II.A). Only when it became possible to critically evaluate protein transport across the endoplasmic reticular membrane of yeasts, as a result of the development of appropriate in vitro systems, did it become apparent that endoplasmic reticulum-targeted proteins such as yeast prepro-a-factor could be targeted to the endoplasmic reticulum in a posttranslational manner (110, 236, 237, 320). This fact led to the suggestion of a unified mechanism for the transport of proteins across cellular membranes (257). Accordingly, interpretation of the SRP-induced elongation arrest as a reflection of a universal mechanism of protein transport was questioned, particularly because the effect could not be shown to occur in all of the in vitro systems tested (175). Clearly, the elongation arrest function of SRP is not a prerequisite for membrane translocation, because these two functions of SRP can be severed biochemically

15 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 347 (272). On the other hand, the translocation-promoting activity appears to be influenced by the arrest activity: a truncated SRP devoid of arrest-mediating proteins exhibited diminished translocation activity unless the membrane concentration was raised (272, 273). In contrast to the native form, the truncated SRP also did not allow for translocation into microsomes that were added late during the reaction (272, 273). Furthermore, extensive investigation of the elongation arrest properties of SRP has revealed that arrest does not always occur at the same position in the nascent chain, as observed for prolactin (316) and immunoglobulin G light chain (174). Interference by SRP of the translation of other proteins occurs at multiple sites in a manner leading to overall retardation of protein synthesis (169). It should be noted that failure to detect an SRP-arrested fragment of a secretory protein does not necessarily mean that SRP has no influence on the translation kinetics. Posttranslational translocation of several proteins across the endoplasmic reticulum has been demonstrated. A closer look at these proteins reveals that they fall into three groups. The members of the first group contain fewer than about 70 amino acids (129, 345). These proteins are likely to be released from the ribosome before recognition by SRP can influence their rates of biosynthesis (see section V.A). The members of the second group remain ribosome bound after termination of translation (40, 43, 178, 200). Possibly their association with the ribosome holds them in a translocationcompetent state. The third group consists of certain yeast proteins targeted to the yeast endoplasmic reticulum (see references 96 and 111 and references therein). The basis for their distinctive characteristics is not known. However, it is clear that posttranslational translocation of polypeptides larger than 70 amino acids, occurring from a ribosome-free precursor pool, has been demonstrated for proteins targeted to the yeast endoplasmic reticulum as well as to mitochondria and the bacterial plasma membrane. The report by Chen and Tai (44), showing that the posttranslational export of bacterial proteins requires hydrolyzable ATP, opened up a new chapter concerning the energetics of protein transport across cellular membranes. On the basis of the finding that proteins which retain a stable tertiary structure are not translocation competent (75, 219), the ATP requirement was hypothesized to be involved in the unfolding of completed precursor chains prior to translocation (238). This idea was subsequently corroborated by demonstrating the ATP dependence of posttranslational transport into the endoplasmic reticulum (110, 200, 236, 237, 261, 320, 328) and mitochondria (74, 205) and by more recent studies concerned with the relationship between protein conformation and translocation competence (49, 50, 73, 208, 301). Subsequently, several proteins have been identified which appear to function in maintaining an unfolded conformation appropriate for transmembrane translocation. These include the heat shock-related proteins from yeasts (51, 64, 186) and reticulocytes (346), which facilitate posttranslational transport into the endoplasmic reticulum and mitochondria. Analogous factors in bacteria were discussed in section IV.A. The recent discovery that one of these bacterial proteins, trigger factor, appears to be replaceable by SRP for protein transport across both the procaryotic plasma membrane (57) and the eucaryotic endoplasmic reticular membrane (253) raises the question of the extent to which SRP, in addition to its various well-defined functions in protein secretion (see section II.A), also functions to maintain the precursor in an appropriately unfolded conformation. This suggested function can also be extended to ribosomes, in the absence of which many proteins do not cross the endoplasmic reticular membrane (see above). Given the facts that proteins can be transported across membranes only in an unfolded state and that the unfolded state is apparently maintained as long as the precursor is still bound to the ribosome, one might assume that cotranslational targeting of nascent chains to membranes should be the prevailing mechanism. However, such temporal coordination might not always be easy to achieve, since translation and membrane translocation are complicated metabolic processes involving a variety of enzymatically catalyzed steps whose net rate depends on a multitude of factors. If, therefore, the translational rate of a given protein exceeds that of its translocation under certain metabolic conditions, mechanisms must be operative which allow the maintenance of transport competence. This could be achieved by the binding of antifolding factors to the growing polypeptide chain. Alternatively, the rate of synthesis could be reduced, as originally postulated for SRP as a consequence of its elongation-retarding activity. Which of these mechanisms predominates may depend on individual characteristics of each precursor, such as its translational rate. This notion implies that not all proteins have the same requirements for insertion. Moreover, it is conceivable that a protein can be processed via different routes, which proceed with different efficiencies (209). Thus, if nascent chains escape one of the coordinating mechanisms just mentioned, the completed polypeptides might be rescued by one of the unfolding (or antifolding) proteins following detachment from the ribosome. Whatever mechanisms might apply, the specificity of the process must ultimately be guaranteed. Thus, an antifolding or unfolding protein may or may not be specific for secretory proteins (see the last two paragraphs of section IV.A), but either it or another protein must ensure that each secreted protein is targeted to the correct membrane site. The specificity of this interaction may partially account for the occurrence of signal sequence-like stretches in proteins (14, 135). The evolutionary pressure for selection of aminoterminal sequences may in part reflect the need to tag the nascent polypeptide chain with a targeting and/or antifolding protein early during its synthesis. Fine tuning of the rate of translation with that of translocation may occur. A large number of suppressor mutants of E. coli have been isolated which partially overcome the export-defective phenotype of seca (157, 191) and secy mutations (269, 270). In general, the suppressor mutations map in genes encoding components of the translation machinery. Moreover, the seca-dependent export defect can be suppressed by sublethal doses of the elongation inhibitor chloramphenicol (157). Conversely, high-level expression of exported proteins, exceeding the export capacity of the cell, induces the synthesis of components of the export machinery (231) and thereby results in the formation of additional intracellular export sites (313). Randall and Hardy (219) proposed that bacterial export proteins partition between a productive transport pathway and a folding pathway which is incompatible with transmembrane transport. In agreement with this suggestion, Liu et al. (170) demonstrated that intragenic suppression of an export-defective signal sequence mutation, owing to a mutation in the mature part of the affected export protein, resulted in retardation of folding and thereby enlarged the time window for proper translocation. A challenging task, therefore, is the elucidation of the mechanisms by which cells of procaryotic origin coordinate protein synthesis with export.

16 348 SAIER ET AL. VII. DISTINCTIVE FEATURES OF MITOCHONDRIAL TARGETING A. N-Terminal Amphipathic Targeting Sequences In contrast to the hydrophobic, endoplasmic reticulumtargeting sequences, mitochondrial targeting sequences do not contain extended stretches of hydrophobic residues. Instead, they possess amphiphilic (amphipathic) character with several distinctive features (233, 258). These features are as follows: (i) the N-terminal signal sequences form an amphiphilic structure, usually an a-helix, with hydrophobic residues on one side of the helix and hydrophilic residues on the other side, but lacking a consensus sequence; (ii) these signal sequences bear a net positive charge, and acidic residues are rare; (iii) hydroxyamino acyl residues are common within these sequences; and (iv) following these sequences are usually (but not always) found cleavage sites, the targets of a matrix-localized processing protease. It is probable that an amphipathic leader sequence as short as 12 residues is sufficient to target a protein to the mitochondrion (126). The compositions of mitochondrial targeting sequences and comparisons with amphiphilic bacterial targeting sequences will be presented in section VIII.C. It should be noted that exceptions to the general description of mitochondrial leader sequences presented above may exist (281), but the recent demonstration that mitochondrial protein import can apparently bypass the need for proteinaceous outer membrane surface receptors (202) shows that caution must be exercised in interpreting the results of import experiments designed to elucidate the structure-function relationships of mitochondrial amphiphilic leaders. Receptor-independent import occurs with low specificity and efficiency and may not require all structural aspects of the leader sequence (205; see Addendum in Proof). Positively charged, N-terminal, amphiphilic a-helices are found with reasonably high frequency in eucaryotic, procaryotic, and viral proteins (246; see section VIII.B). Nevertheless, many of the eucaryotic and viral proteins possessing these structures do not appear in mitochondria. It is probable that such sequences must be suitably exposed on the surface of a protein to allow appropriate interaction with the as yet uncharacterized mitochondrial import machinery. Most soluble enzymes and many integral membrane proteins contain internal amphipathic at-helices with hydrophobic moments of substantial magnitude (M. H. Saier, Jr., and P. McCaldon, unpublished observation). Unmasking of these sequences can confer upon them targeting capacity. For example, the cytoplasmic mouse enzyme dihydrofolate reductase contains an internal, positively charged, amphiphilic ox-helix, which, when placed at the N terminus of two different passenger proteins, targets them to the mitochondrion (127). A masked and nonfunctional amphiphilic sequence can thus be converted into a mitochondrial presequence if appropriately exposed on the protein surface. Comparative studies with bacterial amphiphilic targeting sequences (see section VIII.A) reveal that these N-terminal structures in bacterial permeases are usually punctuated at their C-terminal ends by helix-breaking residues (proline or adjacent glycines). Consequently, the need for exposure may be universal. The possibility that unmasked, internal amphipathic helices function in transmembrane polypeptide translocation will be considered in section VIII.E. MICROBIOL. REV. B. Physicochemical Properties of Mitochondrial Targeting Peptides To gain information about the mechanisms by which mitochondrial targeting sequences interact with biological membranes, the physicochemical properties of synthetic peptides at hydrophobic-hydrophilic interfaces have been examined (35, 36, 232, 234; see section VIII.C). The validity of this approach is suggested by the fact that such signal peptides can inhibit the uptake of authentic mitochondrial precursor proteins into mitochondria in vitro (196, 309). In one careful study (232), three peptides corresponding to the first 15, 25, and 33 N-terminal residues of the subunit IV precursor of yeast cytochrome oxidase were synthesized and studied. Although all three peptides were soluble in aqueous buffer solutions, they spontaneously inserted from an aqueous subphase into phospholipid monolayers. The two longer peptides also caused disruption of unilamellar liposomes in a process which was accelerated by a transmembrane diffusion potential, negative inside. These results showed that the peptides have affinity for phospholipids and that they can traverse and thereby disrupt phospholipid bilayers in a process dependent on charge interactions with a membrane potential. One of the peptides (that of 25 residues) was shown to possess little secondary structure in an aqueous salt solution, but the presence of sodium dodecyl sulfate micelles induced at-helicity to the extent of about 45% (232). The detergent micelles served to provide an appropriate hydrophilic-hydrophobic interface with which the peptide could interact during examination of its spectral characteristics by circular dichroism. These studies revealed that the peptide assumed considerable secondary structure (probably a(-helical) when in contact with the detergent micelles. Unfortunately, no studies were reported that were aimed at determining the degree to which the peptide had saturated the micellar suspension. Since examination of the amino acyl sequence of the signal peptide had revealed that an a-helical configuration was highly asymmetric, with charged residues on one side of the helix and hydrophobic residues on the other side, the induction of a helical configuration was in accord with expectation. Similar results have been reported by other investigators (83, 187). It is clear that amphiphilicity is a crucial factor in mitochondrial targeting and the initiation of membrane translocation, but the relative significance of lipid versus protein interactions to the overall process has yet to be evaluated. Allison and Schatz (7) demonstrated that artificially created presequences composed of only three types of residues (arginyl, seryl, and leucyl residues) could function as efficient import signals in mitochondria. However, these artificial presequences behaved somewhat differently than had been expected: a sequence designed to be weakly amphiphilic as an at-helix proved to function better than one expected to be strongly amphiphilic in an a-helical configuration. This unexpected result cast doubt on the postulate that the amphiphilic structures recognized at the mitochondrial surface must be helical. To directly measure the degrees of amphiphilicity of these leaders in solution, the peptides corresponding to the Arg- Ser-Leu leaders, as well as several other peptides, were synthesized and studied (234). All active presequences were amphiphilic, whereas the inactive presequence studied (which was strongly hydrophilic) was not. One of the active peptides was shown to be nonhelical both in solution and in the presence of detergent micelles. For technical reasons,

17 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 349 the interactions with phospholipid liposomes were not measured. The results substantiate the suggestion that although amphiphilicity is a requirement for presequence activity, an ot-helical configuration is not. The need to consider flexibility of a peptide in assessing its amphiphilicity is suggested by the occurrence of prolyl residues in targeting sequences (99; see section VIII.A). In a recent study (discussed in section VIII.C), Tamm et al. showed that a particular amphipathic peptide may interact with detergent micelles and phospholipid liposomes with quite different degrees of induced helicity (291). It is therefore possible that a helical configuration represents a preferred but not exclusive configuration for active targeting sequences in association with the receptor or lipid constituents in the membrane. The strong tendency of natural, N-terminal, mitochondrial and bacterial targeting sequences to exhibit strong amphiphilicity with large hydrophobic moments when arranged in an a-helix cannot be attributed to coincidence (see also section VIII.A). C. Targeting Information Encoded within the Primary Sequences of Mitochondrial Precursor Proteins A protein targeted to the mitochondrion must choose a suborganellar location: outer membrane, inter membrane space, inner membrane, or matrix. As documented and discussed above, studies with genetically engineered fusion proteins have established the N-terminal amphipathic leader sequence of many mitochondrial precursor proteins to be both necessary and sufficient for targeting of these proteins to the mitochondrion. These amphiphilic leaders do not, however, specify the suborganellar localization. Following the nonhelical, amphipathic leader of the major 70-kDa protein of the yeast mitochondrial outer membrane is an uninterrupted stretch of 19 uncharged, hydrophobic amino acids, flanked on either side by hydrophilic basic residues. This sequence is sufficiently long and hydrophobic to span the membrane as an at-helix and is believed to anchor the protein in the outer membrane (116). Attachment of the N-terminal 61 amino acyl residues of the 70-kDa protein to E. coli P-galactosidase was shown to direct the fusion protein to the mitochondrial outer membrane (115). These results led to the possibility that the 70-kDa protein contains two rather than one targeting signal. The first 12 amphiphilic residues of this sequence apparently represent the first such signal. It merely directs the protein to the mitochondrion; i.e., it is an organellar targeting sequence. If this short sequence is attached to a passenger protein which by itself is not targeted to mitochondria, the resulting fusion protein is transported across both membranes of the mitochondrion to the matrix space (126). Transport to this innermost compartment is thus the ultimate destination of the protein if only this one targeting signal is present. The additional information in the 70-kDa protein appears to be contained within the hydrophobic transmembrane segment (115, 126). It is thought to function as a stoptransfer signal, causing the protein to arrest transfer when associated with the first target membrane, the outer mitochondrial membrane. When this region is altered by deletion of residues within this sequence, the protein does not stop at the outer membrane and instead passes on to the matrix space (258). This situation may prove to be similar to that described below for the mannitol permease of E. coli, from which an amphipathic leader sequence of only 13 amino acyl residues can target a passenger protein to the bacterial envelope fraction and cause it to be translocated across the cytoplasmic membrane. The first hydrophobic segment (of 21 amino acyl residues in the mannitol permease), which is believed to traverse the membrane as a helix, apparently anchors the protein in the inner membrane and prevents further passage to the periplasmic space. The details of this procaryotic system will be discussed in section VIII.D. Targeting of at least some proteins to the mitochondrial intermembrane space is believed to involve an apolar segment which is less hydrophobic than the stop-transfer sequences of integral membrane proteins. Thus, from results of fusion protein studies, residues 1 to 36 of the cytochrome c1 precursor appear to be a matrix-targeting signal, whereas residues 37 to 54 direct the protein to the intermembrane space. This protein, cytochrome b2, and the Fe/S protein of respiratory complex III are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps. The precursors are first translocated across both the inner and outer mitochondrial membranes via contact sites where the two membranes are closely and stably apposed (113, 114, 266). Upon their entry into the mitochondrial matrix, processing by the matrix peptidase generates partially cleaved proteins of intermediate size, which are redirected across the inner membrane by virtue of their newly generated, N-terminal, hydrophobic sequences (113, 114). Only after entry of these proteins into the intermembrane space does the second proteolytic step occur, giving rise to the mature proteins. It appears that in these cases, the hydrophobic stretches in the presequences of the partially processed intermediates act as export signals to direct the exit of the proteins from the matrix into the intermembrane space. Thus, although a hydrophobic sequence apparently serves as a stop-transfer sequence in the 70-kDa protein, different hydrophobic sequences in cytochromes c1 and b2, as well as in the Fe/S protein of respiratory complex III, may serve to reinitiate transmembrane translocation. From the results of work with these and other proteins, it has been proposed that several distinct receptor proteins on the outer surface of the outer mitochondrial membrane recognize specific precursor proteins and transfer them to the common constituents of the protein translocation machinery in the outer membrane (203, 206). The import pathways then diverge either to the outer membrane (as for the 70-kDa protein) or to translocation contact sites (as for the cytochromes discussed above). In the latter case, further divergence is responsible for directing these matrix-targeted proteins to their final mitochondrial compartments. VIII. DISTINCTIVE FEATURES OF BACTERIAL PTS PERMEASE INSERTION The PTS permeases, each of which is specific for a particular sugar, possess characteristic features: all are integral proteins of the bacterial cytoplasmic membrane which possess about 51% hydrophobic residues, 31% semipolar residues, and 9% each of the acidic and basic residues. The vast majority of these proteins possess N-terminal domains which are embedded in the membrane and probably span the membrane as a-helices 7 to 10 times. The C- terminal domains usually exhibit the properties of typical soluble proteins and are localized to the cytoplasmic side of the membrane. Although some of these permeases exist as a single polypeptide chain (enzymes II), others consist of two chains (enzyme II-III pairs). The sizes of these permeases are relatively invariant (for comparative structural analyses, see references 245a, 247 and 248).

18 350 SAIER ET AL. Sequence comparisons of these permeases revealed that although many of them exhibit little sequence identity throughout their lengths, almost all of them possess N- terminal, amphipathic, potentially helical leader sequences with exclusively hydrophobic residues on one side of the helix and exclusively hydrophilic residues on the other side (249). This observation and a knowledge of the significance of similar sequences to mitochondrial targeting led to the postulate that these structures were of biogenic importance. They also led to the studies reported below, which establish them as bacterial envelope-targeting sequences. Such a leader sequence not only targets a newly synthesized protein to the cytoplasmic membrane but also appears to direct the initiation of its translocation through the membrane. A. N-Terminal, Amphipathic Leader Sequences As discussed in section VII, a short, N-terminal amphipathic leader sequence, properly situated in a eucaryotic protein, is both necessary and sufficient for import of that protein into mitochondria. In most cases, the leader sequences are capable of forming amphipathic ox-helices with hydrophobic moments of large magnitude. Attachment of such leaders to the N termini of various proteins results in their binding to the outer mitochondrial membrane (102) and import into mitochondria (126). Since bacteria lack organelles such as mitochondria, some investigators have assumed that mitochondrial assembly processes are eucaryote specific and that they evolved after the divergence of eucaryotes from procaryotes (14). However, it is the thesis of this review that molecular processes occurring in eucaryotes frequently find their parallel in procaryotes. Consequently, protein targeting and membrane insertion in the two major groups of living organisms may occur by fundamentally similar processes using similar targeting signals. In this section we summarize evidence which favors this notion with respect to the use of amphiphilic helices as targeting sequences. The sugar recognition components of the bacterial PTS, the membrane-embedded enzymes II, serve as sugar permeases, kinases, and chemoreceptors (212, 244). Although the mannitol enzyme II (enzyme 11MtI) of E. coli was the first PTS permease for which the primary structure became known (159) and is still the best characterized of this class of permeases, recent gene sequence work has allowed deduction of the amino acyl sequences of 13 of the enzymes II from various bacterial species. The primary structures of the N termini of 11 of these 13 enzymes II are shown in Table 2. All of these 11 sequences were found to be capable of forming amphipathic leader a-helices with exceptionally large hydrophobic moments (76, 77, 84, 249; M. H. Saier, Jr., unpublished observations). Each of the PTS leader sequences is terminated by at least one a-helix breaker, a prolyl or two adjacent glycyl residues, although sometimes the helix breaker occurs toward the end of the amphipathic region. This observation suggests that the leader sequence might function independently of the rest of the protein. Axial projections of potential ot-helical conformations of the N-terminal sequences of eight enzymes II of the PTS have been published (249). These leaders were 13 to 21 amino acyl residues long (Table 2), with essentially all of the amino acyl residues on one side of the helix exhibiting hydrophilic character and all residues on the opposite side of the helix exhibiting hydrophobic character. With only a few minor exceptions, no hydrophobic residues were found on MICROBIOL. REV. the hydrophilic sides of these helices and no hydrophilic residues were found on the hydrophobic sides. The sequences of three sucrose enzymes II and three lactose enzymes II from a variety of bacterial species are now available (Table 2). The leader peptides of the three sucrose enzymes II are shown in helical wheel projection in Fig. 3, and those of the three lactose enzymes II are shown in Fig. 4. The amphiphilicity of these helices is apparent. It is worth noting that there is minimal sequence identity between these sequences (Table 2), even though the three proteins specific for each of the two sugars show about 50% sequence identity throughout the majority of their lengths. This fact argues in favor of evolutionary pressure for the retention of amphiphilicity without retention of any specific sequence. The fructose enzyme II is atypical among PTS permeases in that the hydrophilic portion of the molecule (residues 1 to 229) precedes the hydrophobic portion (residues 230 to 563) (213a). This is contrary to the pattern seen for all other enzymes II studied (see, for example, references 213a, ). In agreement with this fact, no amphipathic signal sequence was found at the N terminus of this enzyme. Interestingly, however, two amphipathic a-helices were found to precede the first presumed transmembrane hydrophobic sequence (Fig. 5). The possibility that one or both of these internal amphipathic a-helices function to initiate protein insertion will be discussed in section VIII.E. In view of the excess of positive charge found within the leader presequences of virtually all imported mitochondrial proteins (67, 232, 309), it is interesting that although most enzymes II possess leader sequences with an excess of positive charge, enzymes IIScr-2 and IlFru possess neutral leader sequences, and enzymes ligut, iiscr-1, and IIScr-3 possess leader sequences with a net negative charge. Amino acid compositional analyses of residues 2 to 12 of the 11 N-terminal leader sequences shown in Table 2 are summarized and compared with those of mitochondrial leaders in Table 3. The results reveal that prolyl, cysteyl, and glycyl residues are rare, consistent with an a-helical configuration. Isoleucyl, phenylalanyl, glutamyl, and lysyl residues show increased frequency relative to their average occurrences in proteins. The enzyme IlFru leader also shows this compositional tendency. These compositional differences will be discussed further in section VIII.C. As noted above, the 11 enzyme II leaders shown in Table 2 are each terminated by at least one ax-helix breaker, either a prolyl residue or two adjacent glycyl residues. Two nearby helix breakers sometimes terminate the leader sequence. The fact that both helical and nonhelical amphipathic leader presequences of 12 or more residues can effect the import of proteins into mitochondria (7, 309) may be relevant to these enzyme II leader sequences. The N-terminal sequences of enzymes II_pMan and II_MMan, the two subunits of the structurally distinct mannose enzyme 11 (85), were found to exhibit none of the features characteristic of the other enzyme II leader sequences. The N-terminal 18 amino acyl residue sequences of numerous membrane or membrane-associated bacterial proteins were examined for their hydrophobic moments and average hydrophobicity values (76, 77, 249). Among the proteins examined were proton- and sodium-carbohydrate symport permeases of E. coli, periplasmic-binding proteindependent permease components of E. coli and Salmonella typhimurium, components of the FO-ATPase of E. coli, several pigment-binding proteins of Rhodobacter capsulatus, electron transfer components of E. coli, and a variety of

19 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES t 30. C-3. -X U) 0 0_ 0 0 C Do r r 0 3 A. fo DCC.r-. 0 ~ C). 0 DC W 0 (1 0) e0 r ro r 4 > zo,,o== o r + ~~~~I) ~I IC Z - (IQI C H rtl 0 D ~ < C 5. Z. ~ r roll 0 0)" + r) > + +: I z 0 C 00 t C. C) 0. 0 _~. Ui ni + < rez> nil + -r-2 + +TI + + It :>g -e r- It 0- ;t+ C _I Di 0< nitl + -4 o cn< rl, > C) > - 0- l+ 1+ C)I > 0 Q r +q +;l C t* Itm ozc + ;V w C)~T -r z P > o DC ~ Ci) w.. ro 0 (D a) z 0) 0 0- DC 0 5. b 10 PD 0(A (DC 0. 0 C) 0 El tt Unz DC co _.00 C3 la ~0 U0 0 cn_ Li) -- O tj 00 I- wb w (IQ =ri P) >C 5 - PDeO g H (D. D e.=u W =r* O - 0 Br

20 352 SAIER ET AL. MICROBIOL. REV. WI A A S 7 L 14 S C E) M I E>' If p 12 Q?_5 E strongly amphipathic leader presequences of imported mitochondrial proteins (249). These observations lead to the suggestion that the presence of the amphipathic leader sequences at the N termini of the PTS enzymes II must have been selectively advantageous throughout evolutionary history and must therefore serve an important function. B. Statistical and Functional Analyses of Proteins with N- S R Terminal, Amphipathic, Potentially a-helical Sequences Although overwhelming evidence had long supported a role of N-terminal amphipathic a-helical sequences in mitochondrial targeting in eucaryotes, no detailed computer 2 00 ( analyses had, until recently, been conducted to ascertain the t=>0@ (2> statistical or functional significance of such structures in [3 proteins. Consequently, Saier and McCaldon (246) under- L took an analysis of nearly 2,000 proteins with N-terminal T 0i) methionine (presumably nonprocessed) and, separately, al- 3 T most as many presumably processed proteins with other F 10 Ea y L IYi amino acids in their N-terminal positions. The proportions of N-terminal sequences, 18 residues in length, with a-helical hydrophobic moments of various values (76) were the same within the limits of experimental FIG. 3. a-helical wheel projection of the: N-terminal leader error for the two classes of proteins. When plastid (mitosequences of the three sucrose enzymes II. Thes;e are the enzymes chondrial and chloroplast) and viral proteins were deleted from S. typhimurium (probably originally from a JKlebsiella sp. [71]) from the list, only 3.5% of the proteins exhibited leader (letters nearest the wheel), B. subtilis (92) (next Iletters out), and S. sequences with hydrophobic moment values per amino acyl mutans (256) (letters furthest from the wheel1). The one-letter residue abbreviations of the amino acids are used. The d ((RH) values) in excess of 0.4. By contrast, about the division between the hydrophobic and hydrojphilic piagonal halves line shows of the 10% of both viral and membrane proteins exhibited (RH) helices. values in excess of 0.4. Each protein with a large hydrophobic moment and known function was categorized and statistically analyzed. Of the proteins with known functions, periplasmic and outer membrane proteins )f gram-negative about 55% interacted functionally with nucleic acids, 30% bacteria. With only one exception (the rb,,sd protein), the were membrane-interacting proteins or their precursors, and hydrophobic moments of all of these N-teriminal sequences about 15% were viral structural proteins, primarily confell within the normal range (0 to 0.3) (24' 9; Saier, unpub- cerned with host-cell interactions (246). The majority of lished observations). It is interesting that the N-terminal these sequences possessed a net positive charge. A striking leader sequences of bacteriorhodopsin and halorhodopsin example of a protein with a most unusual N-terminal ams and that these phipathic a-helix (21 residues in length) was the host speci- can be drawn as weak amphipathic helice, leaders have been postulated to facilitate rmembrane inser- ficity protein B of phage T7, in which all hydrophilic residues tion (311). on one side of the helix were lysyl residues and all hydro- sequences phobic residues on the other side were valyl residues (246). Hydrophobic moments of the enzyme II Ileader are similar in magnitude to those of the amiphipathic, mem- The observations reported suggested that N-terminal am- the S. aureus phipathic a-helices, particularly of viral proteins but also of brane-disrupting, a-helical toxins such ass hemolysins, artificial cytotoxins, and the melittins (76). cellular proteins, function in nucleic acid binding, facilitate Moreover, they are comparable in magnitiude to the most membrane insertion, and promote virus-host cell interac- M B L M I e I F 12 F >, 5 C I I1I A 6 i9 L 3 ii to 01 2 T ' 13 6 K K p FIG. 4. Leader sequences of the three sequenced lactose enzymes II. These enzymes were from S. aureus (A), Lactobacillus casei (B), and S. lactis (C). The conventions used were the same as for Fig. 3. Note that the borders between hydrophobic and hydrophilic sides of the three helices are in different positions. L 6 p E(3 2 He

21 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 353 K ( L K ( I I S Q V L R 18 A S A FIG. 5. Helical wheel projections of the two amphipathic segments of the fructose enzyme II (residues 188 to 203 and 211 to 228) preceding the first presumed transmembrane hydrophobic helix (starting at residue 230). The short loop connecting the two amphipathic segments is also shown. Amino acid I V L. F M A PI G w T S Y J Q N E DJ H K R Totals TABLE 3. Amino acid compositions of leader sequences from nucleus-encoded, mitochondrion-targeted precursor proteins of eucaryotes and envelope-targeted PTS-permease proteins of procaryotes Amino acid class Hydrophobic residues Helix breakers Hydroxy residues Acidic residues Basic residues Hydrophobic residues Helix breakers Hydroxy residues Acidic residues Basic residues Bacterial targeting sequences in PTS permeases' T T T T T 17.4T Eucaryotic mitochondrial targeting sequencesb 3.01 d t t 11.1T T 51.2T T T Relative abundance in proteins' 5.7d Residues 2 to 12 inclusive of the 11 sequenced enzymes II of the PTS which show large hydrophobic moments for their N-terminal leaders were used for the compositional analyses reported (Table 2). Symbols:, significantly increased frequency of occurrence of this residue relative to its occurrence in proteins in general; I, significantly decreased frequency of occurrence of this residue relative to its occurrence in proteins in general. b Data were taken from reference 307. Symbols are as in footnote a. c Data were taken from reference 245. d Percentage of total. tions. These structures therefore appear to serve the generalized function of macromolecular recognition. C. Physicochemical Properties of PTS Targeting Peptides and Comparison with Mitochondrial Targeting Peptides Studies concerned with the interactions of a variety of peptides, including signal peptides, with artificial and biological membranes have recently been comprehensively reviewed (135). Hydrophobic leader peptides corresponding to the signal sequences of both eucaryotic and procaryotic secretory proteins have been studied. Of the bacterial leader peptides, those corresponding in sequence to the leaders of the M13 coat protein, the outer membrane maltoporin (LamB), and the outer membrane anion porin (PhoE) have probably been the most extensively studied (19, 135). Amphiphilic leader peptides of nucleus-encoded mitochondrial precursor proteins have also been synthesized and studied by physicochemical techniques (7, 187, 232, 234, 280, 290; see also section VII.B). Until recently, no corresponding studies with synthetic amphiphilic peptides corresponding to targeting sequences of bacterial origin had been made. Such studies were considered to be important because the bacterial targeting sequences differ in several interesting respects from the mitochondrial targeting sequences. First, although both structures usually show strong amphiphilicity when arranged in an a.-helix, the bacterial leaders can possess either a net positive or a net negative charge (Table 2), whereas mitochondrial leaders virtually always possess a net positive charge (as discussed in section VII.A). Second, although hydroxyamino acyl residues occur with high frequency in mitochondrial leaders, they are present in the leaders of PTS permeases with depressed frequency (Table 3). Third, although acidic residues are extremely rare in mitochondrial targeting sequences, they occur with normal frequency in the PTS leaders (Table 3). Fourth, although both classes of leaders show an increased proportion of hydrophobic and basic residues, the predominant hydrophobic residue is leucine for the eucaryotic leaders, but isoleucine and leucine are present in the procaryotic leaders with equal frequency. The predominant basic residue in the mitochondrial leaders is arginine, but it is lysine for the bacterial leaders (Table 3). Finally, although most eucaryotic

22 354 SAIER ET AL. leaders of mitochondrially targeted proteins are cleaved following translocation, the PTS permease leaders are apparently not cleaved. It is interesting that although both the eucaryotic and the procaryotic amphipathic leader sequences show a decrease in the frequency of helix-breaking residues, this decrease is more pronounced for the bacterial targeting sequences. This last observation may suggest that requirements for a helical leader are more stringent in bacteria than in eucaryotes. The earlier studies on mitochondrial targeting peptides (see section VII.B) had shown that signal peptides of cytochrome c oxidase, subunit IV, and sorne artificial mitochondrial targeting sequences exhibited amphiphilic a-helical or n-strand structures when associated with detergent micelles or phospholipid vesicles (232, 234). It was shown that these and some other mitochondrial signal peptides were capable of inserting efficiently into preformed phospholipid monolayers and bilayers in a process which depended on both hydrophobic and charge interactions (187, 280, 290). These studies led to some proposed modes of membrane association and insertion. In a recent study, amphipathic peptides corresponding to the N termini of two PTS permeases, the glucitol enzyme II (enzyme ligut) and the mannitol enzyme II (enzyme llmtl), were synthesized and their interactions with phospholipids and detergents in various hydrophobic-hydrophilic interfaces were studied (291). Both peptides were found to insert into phospholipid monolayers, and in agreement with results of the earlier studies, their capacities to partition into monolayers correlated with both their lengths and their average hydrophobicities. Thus, the glucitol enzyme II leader peptide, Gut-22, which exhibited substantially greater hydrophobicity and was longer than the mannitol enzyme II leader peptide (Mtl-15), incorporated to a greater extent into the monolayers than did Mtl-15. Circular dichroic analyses revealed that association of the peptides with appropriate model membranes induced secondary structure. In Gut-22, this secondary structure was almost entirely a-helix. Up to 70% of the peptide appeared to assume an a-helical configuration. Furthermore, tryptophan fluorescence measurements with Gut-22 confirmed the conclusion that the peptide became embedded in the hydrophobic membrane interior. These results gave experimental support for the postulate that the N-terminal signal sequences of the PTS permeases form amphiphilic secondary structures in the presence of a hydrophobic-hydrophilic interface. They support a targeting mechanism in which the hydrophobic face of the amphiphilic helix associates with a hydrophobic surface, either phospholipid or protein. The a-helix could insert parallel to the plane of the membrane (in which case a surface location is implied) or perpendicular to it (in which case association with a hydrophilic moiety of another protein moiety is implied). It should be noted that neither of these two possibilities excludes the other, since both orientations might function in sequence in the insertion process. D. Targeting Conclusions Resulting from Studies with Fusion Proteins Recent experiments have provided clear evidence for an essential role of the N-terminal amphipathic leaders of PTS permeases in envelope targeting (Y. Yamada, M. Yamada, and M. H. Saier, Jr., unpublished results; Daniels and Saier, unpublished). Thus, by using the technology of Manoil and Beckwith (172), Boyd et al. (29), and Boquet et al. (26), two fusion proteins were generated in which N-terminal amphi- MICROBIOL. REV. TABLE 4. Distribution of alkaline phosphatase activity between the inner and outer membranes of E. coli strains which synthesize the F13 and F53 MtlA-PhoA fusion proteins Membrane Succinate KDa Alkaline Strain Mfractbion e dehydrogenase (plmol/ml) phosphatase Strain raction(pmol/min per ml) (/l LJ922(pMA28) Inner LJ922(pMA28) Outer LJ922(pMA27) Inner LJ922(pMA27) Outer a KDO, 3-Deoxy-D-manno-octulosonate. pathic sequences derived from enzyme llmt' were fused to the functional moiety of alkaline phosphatase. The two fusion proteins, which were characterized by genesequencing techniques, had the N-terminal signal sequence of alkaline phosphatase (including the cleavage site) replaced with either the N-terminal 13-amino-acyl leader sequence of enzyme llmtl (fusion protein F13 encoded by plasmid pma28) or the N-terminal 53-aminQ-acyl residues of enzyme llmtl (fusion protein F53 encoded by plasmid pma27) (Yamada et al., unpublished). The cellular locations of the two hybrid proteins F13 and F53 were determined by cell fractionation. Comparison of total activity in the cell extracts with that in intact cells revealed that virtually all of the activity was extracellular. None of the activity was released by osmotic shock of intact cells, however, suggesting that the fusion proteins were not soluble in the periplasm but were membrane bound. Cell disruption by passage through a French pressure cell released in a soluble form a majority of the activity encoded by plasmid pma28 but much less of that encoded by pma27. The remainder of the activity remained in the particulate fraction and could be sedimented by high-speed centrifugation. Table 4 summarizes the distribution of alkaline phosphatase and representative markers of the inner membrane (succinate dehydrogenase) and outer membrane (3-deoxy- D-manno-octulosonate) after separation of vesicles derived from the two membranes (Yamada et al., unpublished). As revealed by the data obtained for succinate dehydrogenase and 3-deoxy-D-manno-octulosonate, the two membranes were adequately separated. Particulate alkaline phosphatase activity derived from pma28, associated with the fusion protein containing only the 13-amino-acyl leader sequence of enzyme IlMt, was distributed between the two fractions, with about 60% of the activity associated with the outer membrane. By contrast, activity derived from pma27, associated with the fusion protein containing 53-amino-acyl residues of enzyme llmt', was associated predominantly with the inner membrane. The larger of the two fusion proteins, F53, contained the amphipathic leader sequence of enzyme llmti plus a hydrophobic stretch of 21 amino acyl residues (residues 24 to 44) (159), which, according to hydropathy analyses (153), is sufficiently long and hydrophobic to pass through the membrane as an a-helix. Since a 53-amino-acyl segment obviously carries more information than a 13-amino-acyl segment, it appeared that the short, amphipathic, N-terminal, 13-residue segment of enzyme limt' must merely target the protein to the envelope fraction of the cell and facilitate passage of the protein through the cytoplasmic membrane. The subsequent sequence of 21 hydrophobic residues (residues 24 to 44) presumably prevented the protein from passing through the inner membrane to a more external

23 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 355 structure (the periplasm, outer membrane, or extracellular fluid). The amphipathic leader sequence therefore appears to target the protein to the envelope fraction and allow transmembrane translocation without specifying its final destination, whereas the first transmembrane (x-helix serves as an anchoring signal to anchor the protein to the inner membrane. The situation proposed here for enzyme 11Mt' of E. coli is highly reminiscent of the situation described in section VII.C for the 70-kDa mitochondrial outer membrane protein of S. cerevisiae. Enzyme IIMt' has a true molecular mass of 68 kda, but, like many integral membrane proteins, it migrates in sodium dodecyl sulfate-gels with a greater mobility than expected on the basis of its size (159). Its mobility is that of a protein with an apparent molecular mass of 60 kda (133). It consists of an N-terminal membrane-embedded domain and a C-terminal domain exhibiting the properties of a water-soluble protein. The latter domain is localized to the cytoplasmic side of the membrane (132, 284). Reciprocal-type fusions, in which the amphipathic (11-residue) leader of enzyme 1jMtl was removed from the enzyme and replaced by a short hydrophobic sequence (the N-terminal 7 amino acyl residues of 3-galactosidase), gave rise to a protein which proved to be inactive, most probably because the permease protein could not be properly inserted into the cytoplasmic membrane (Daniels and Saier, unpublished). Western blot analyses of the mutant enzyme jjmtl lacking its N-terminal amphipathic leader sequence revealed that the mutant protein had been cleaved to a membrane-associated 39-kDa fragment (presumably derived from the N-terminal portion of the enzyme) and a water-soluble 28-kDa fragment (presumably derived from the C-terminal portion of the enzyme). By contrast, chromosomally encoded enzyme IIMtl yielded only one cross-reacting protein with the expected apparent molecular mass of 60 kda in wild type cells. It is reasonable to suppose that exposure of a protease-sensitive site and cleavage of the fusion protein by an endogenous protease resulted from the improper insertion of enzyme llmtl into the membrane because it lacked its N-terminal targeting sequence. Further experiments were carried out with temperaturesensitive seca and secy mutants. When the mutants were induced for the synthesis of the chromosomally encoded enzyme llmtl at the permissive temperature (30 C), only the 60-kDa protein was made. By contrast, the 39- and 28-kDa fragments noted above were observed after extended incubation of either the seca or the sec Y mutant cells (but not of the wild-type cells) at the nonpermissive temperature (42 C). Moreover, overproduction of native enzyme llmt' in wildtype cells gave rise to the two cleavage products in addition to the 60-kDa protein. It had been shown previously that overproduction of the mannitol, glucitol, or lactose permease of E. coli gave rise to inhibition of septation (snake formation) followed by cell lysis (160, 336), suggesting that the insertion machinery could be easily saturated. From these results, it was concluded that loss of the leader sequence of enzyme llmtl, loss of either SecA or SecY protein function, or saturation of the insertion machinery of the cell with newly synthesized enzyme llmt' resulted in improper insertion of the protein into the membrane. Cleavage of the protein to the 39- and 28-kDa fragments therefore appeared to serve as an assay for improper insertion. As noted above, PTS permeases are unusual in possessing N-terminal, amphipathic, oa-helical leader sequences which apparently function in envelope targeting and membrane insertion. Since a few other membrane and secreted proteins have recently been found to possess such amphipathic N termini (for example, bacteriorhodopsins and halorhodopsins of archaebacteria [311] and the PF3 phage coat protein rsee Addendum in Proof]), it is possible that the use of amphipathic at-helices to target proteins to the insertionsecretory apparatus of the cell is not an exclusive characteristic of the PTS permeases in bacteria. Further work is required to establish the general significance of these structures to membrane biogenesis. E. Internal Amphipathic oa-helices as Potential Translocation Signals As discussed in section VIII.A, all of the structurally similar sequenced PTS permeases, with the exception of the fructose enzyme II, possess largely hydrophobic N-terminal domains and hydrophilic C-terminal domains (245a, 249). The fructose enzyme II shows the opposite orientation (213a), and, unlike the other enzymes II, it does not possess an N-terminal amphipathic (x-helix. Examination of the primary structure of this protein revealed that preceding the first hydrophobic transmembrane segment are two segments which are capable of forming ot-helices with fairly large hydrophobic moments (segments 188 to 203 and 211 to 228 [Fig. 5]). Segment 188 to 203 most resembles the other PTS leader sequences, and its linear structure is presented at the bottom of Table 2. The two amphiphilic segments depicted in Fig. 5 are separated by a 7-residue, nonhelical linker region containing two prolines and a glycine. It is possible that one or both of these amphipathic structures serve as an internal targeting sequence to initiate insertion of the growing polypeptide chain into the membrane. Unlike the glucose, N-acetylglucosamine, and glucitol enzymes II, the sucrose enzymes II from enteric bacteria, B. subtilis, and Streptococcus mutans, as well as the 3-glucoside enzyme II of E. coli, have hydrophilic 70- to 100- amino-acid stretches before the first transmembrane segment (245a, 247, 248). This difference most probably arose by intragenic transposition as discussed previously (31, 248). Although these enzymes all possess N-terminal amphipathic sequences capable of forming helices of large hydrophobic moments, immediately preceding each of the first hydrophobic stretches in these four enzymes II there are weak amphipathic sequences. It is possible that these secondary amphipathic segments also play a role in membrane insertion. It is interesting in this connection that Horabin and Webster (121) have recently identified a 55-amino-acyl sequence in the gene I protein of the filamentous E. coli phage fl which directs membrane insertion and causes loss of membrane potential. The active moiety includes a 20-aminoacyl hydrophobic region presumed to span the membrane as an cx-helix, preceded by a 13-amino-acyl sequence which is capable of forming a positively charged amphipathic (x-helix. This internal segment (residues 241 to 295 in the 348-residue protein), when present in a protein, results in insertion of the protein into the bacterial membrane, with sequences on the carboxyl side of the hydrophobic region being translocated across the membrane. These observations are fully consistent with a generalized function of the amphipathic segment in the initiation of transmembrane polypeptide translocation. The recent demonstration that the noncleavable C-terminal signal sequence, directing export of the E. coli hemolysin protein across the bacterial envelope, contains a potential 18-amino-acyl amphiphilic a-helix (141) further supports the notion that internal amphipathic helices may function in membrane targeting and translocation.

24 356 SAIER ET AL. TABLE 5. Alkaline phosphatase activities associated with four -MtlA-PhoA fusion proteins Fusion Mannitol Alkaline proteina Plasmid fermentationb phosphatase F13 pma28-10,500 F53 pma27-21,900 F432 pma F465 pma30 d 11,100 a The number in each subscript specifies the number of amino acyl residues derived from enzyme II"M which are attached to the catalytic domain of alkaline phosphatase via a 2-kDa linker (Yamada, et al., unpublished). The procedure of Manoil and Beckwith (172) was used for their construction. b Fermentation was measured on eosin-methylene blue plates containing 0.5% mannitol. c Specific activities are expressed in nanomoles of p-nitrophenol formed from p-nitrophenyl phosphate per minute per milligram of protein at 37 C (172). d Fermentation was substantially weaker than for the wild-type strain. Complementation with another enzyme II provides the probable explanation for the positive fermentation response of this strain (302; S. Sutrina and M. H. Saier, Jr., unpublished results). Recently, two fusion proteins of the mannitol enzyme II and alkaline phosphatase (mtla-phoa gene fusions) were constructed which may be relevant to the possible role of internal amphipathic ox-helices in polypeptide translocation (Yamada et al., unpublished). Sequence analyses of these fusion proteins revealed that the shorter of them, F432, encoded within plasmid pma24, contained 432 amino acyl residues derived from enzyme IIMt1, whereas the longer one, F465, encoded on plasmid pma30, contained 465 residues derived from enzyme IlMt'. The two fusions exhibit a difference in length of 33 amino acids. The last presumed transmembrane, hydrophobic segment in enzyme llmt1 terminates at residue 334, and the entire remaining C-terminal portion of the polypeptide chain, which exhibits the hydrophobicity properties of a watersoluble protein (159) and can be released from the membrane following mild trypsin treatment as a fully water-soluble fragment, has been shown to be localized to the cytoplasmic side of the membrane (132, 133, 284). When the catalytic moiety of alkaline phosphatase, fused to any protein, is localized to the cytoplasm it exhibits no activity, but when it appears on the periplasmic side of the inner membrane it exhibits full or nearly full activity (5, 26, 28, 29, 118, 172, 173). No exceptions have yet been documented (D. Boyd, personal communication). The shorter of the two MtlA-PhoA fusion proteins, F432, was found to exhibit no alkaline phosphatase activity, as expected, since the portion of the mannitol enzyme II to which the alkaline phosphatase moiety had been fused had a cytoplasmic location (Table 5). However, it came as a major surprise that fusion protein F465 exhibited a high degree of alkaline phosphatase activity, over half the activity of the F53 fusion discussed in the previous section (Yamada et al., unpublished results; Table 5). Examination of the 33-amino-acyl residue sequence between the two sites of fusion (residues 432 and 465) revealed that residue 432 is in the middle of a long, amphipathic, potentially a-helical region (residues 425 to 441) bordered on either side by prolyl residues (residues 421, 422, and 442) (Fig. 6). It is therefore possible that construction of fusion protein F465 unmasked this internal amphipathic helix so that it became recognized as a site of transmembrane translocation initiation. Fusion with the phoa gene in the middle of the corresponding gene segment would, of course, be ex- V gd S - ne3 ue D 1 ARO I R ( t t2 T 10 A I V T V MICROBIOL. REV. FIG. 6. Helical wheel projection of the internal amphipathic segment between residues 425 and 441 inclusive in enzyme 11Mt' (159). This structure is postulated to provide an explanation for the alkaline phosphatase activity of the MtlA-PhoA fusion protein, F465, by serving as an internal targeting sequence (see Table 5 and text). pected to abolish any such function. Thus, the lack of alkaline phosphatase activity associated with F432 is in full accord with expectation. It is, of course, possible that the amphipathic segment derived from enzyme llmt' functions in conjunction with a sequence in the mature portion of alkaline phosphatase. It is important to point out that the results reported above for fusion proteins F432 and F465 show that studies with alkaline phosphatase fusion proteins can reveal topological features of proteins which incorrectly suggest membrane targeting functions in the native protein. Thus, creation of a fusion protein may unmask potential sequences which assume functions dissimilar to those that operate in the native protein. It is clear that caution must be exercised in interpreting the results of alkaline phosphatase fusion experiments in terms of the topographies of integral membrane proteins. IX. UNIFIED MODEL OF PROTEIN INSERTION The results summarized in this review provide new evidence for a single, unified mechanism by which proteins are inserted into and translocated across biological membranes. It is now clear (i) that procaryotes and eucaryotes insert proteins into their membranes and secrete proteins across their membranes by very similar mechanisms; (ii) that both groups of organisms recognize both hydrophobic and amphiphilic leaders as translocation signals; (iii) that both procaryotes and eucaryotes possess a variety of functionally overlapping receptors, with antifolding or unfolding activities, which feed into a common secretion-insertion pathway; (iv) that although bacteria insert and secrete proteins into and across the cytoplasmic membrane by using a variety of receptors, essentially the same mechanism, involving some common components of the insertion-secretion machinery (SecA and SecY), is used; and (v) that the same machinery is used regardless of whether the proteins have hydrophobic (endoplasmic reticulum-targeting-like) leaders or am-

25 VOL. 53, 1989 INSERTION OF PROTEINS INTO BACTERIAL MEMBRANES 357 phiphilic (mitochondrion-targeting-like) leaders and regardless of the target membrane. By extrapolation, it seems likely that the insertion mechanisms for proteins targeted to the endoplasmic reticulum or mitochondrion of a eucaryotic cell will prove to possess similar features. Finally, we predict, on the basis of the results summarized, that appropriate hydrophobic and amphipathic sequences can initiate polypeptide translocation, regardless of whether they are N terminal or internal. It is clear, however, that proper exposure of these segments to a membrane receptor is required for the transport function to be realized. What might the translocation mechanism involve? Reasonably detailed models have been proposed by various workers. We subscribe in principle to the main features of the models elaborated by Blobel and Dobberstein (23), Randall et al. (221), and Singer et al. (278, 279), in which water-soluble protein constituents of the insertion-secretion machinery facilitate transmembrane translocation by controlling the activity of a channel which can accommodate both hydrophilic and hydrophobic moieties of a partially folded but largely denatured protein. The channel must be capable of accommodating secondary but not tertiary protein structures, but it can never be truly open, since such a configuration would result in the free flow of protons and other ions and hence the rapid collapse of the transmembrane potential. Until recently, the functional identification of the specific proteins which make up the translocation machinery had been extensively pursued only for the endoplasmic reticular system of eucaryotes. In the last few years, however, the accumulation of evidence concerning the functions of SecA, SecY, SecB, trigger factor, and GroEL, as well as the most recent evidence suggesting that a soluble export factor contains SecB and might function as the E. coli equivalent of a signal recognition particle (318), indicates that knowledge concerning the bacterial insertion-secretion system will soon outstrip that of the eucaryotic systems, if it has not already done so. Thus, establishment of the coupling of the ATPase activity of SecA to its association with other components of the translocation complex and evidence concerning the topologies of SecY and leader peptidase represent advances yet unsurpassed with eucaryotes. The evidence currently available clearly suggests that the fundamental mechanisms of protein targeting, secretion, and incorporation into biological membranes are similar in all living organisms. Not only are functional parallels apparent, but also structural homologies between components of the insertion-secretion machinery found in eucaryotes and procaryotes are now being revealed. The occurrence of both hydrophobic and amphipathic leader sequences in proteins targeted to the membranes of bacteria and nucleated cells further emphasizes the structure-function parallels. In view of the greater ease of physiological, genetic, and biochemical manipulation in procaryotes relative to any eucaryotic system, the initial lead held by eucaryotic biologists studying membrane biogenesis must have reflected an unfortunate misappropriation of funds and effort for scientific achievement. Assuming the rational natures of scientists and the scientific process, and recognizing the major deficiencies in our understanding of universal biological processes, we can anticipate reversal of current funding policies, a shift in effort toward procaryotic systems, and reestablishment of the earlier trend in which the major basic advances were made with procaryotes. This expectation also follows from recognition of the facts that every life-endowing quality must be shared by all living organisms and that the organisms which are most amenable to detailed experimental analysis are the logical objects of study. ACKNOWLEDGMENTS We thank D. W. Andrews, P. J. Bassford, Jr., J. Beckwith, Y.-Y. Chang, G. A. Daniels, K. Ito, A. Kuhn, C. A. Kumamoto, W. Neupert, D. B. Oliver, A. P. Pugsley, L. L. Randall, M. J. Rindler, B. Rippon, D. Roise, R. Schekman, M. Schwartz, T. J. Silhavy, C. Stirling, P. C. Tai, G. von Heijne, W. Wickner, M. Yamada, Y. Yamada, and R. Zimmermann for providing reprints, manuscripts, information concerning unpublished experiments, and helpful suggestions. Mary Beth Hiller provided expert and conscientious assistance in the preparation of this manuscript. The work reported from our laboratories was supported by Public Health Service grants 5ROlAI21702 and 2ROlAI14176 from the National Institute of Allergy and Infectious Diseases (to M.H.S.) and grants from the Sonderforschungsbereich 206 and the Fonds der Chemischen Industrie (to M.M.). ADDENDUM IN PROOF Work in P. Walter's and B. Dobberstein's laboratories has resulted in the sequencing of cdna clones encoding the 54-kDa subunit of the mammalian signal recognition particle (SRP; see section II A) which is known to interact with signal sequences as they emerge from the ribosome [H. D. Bernstein, M. A. Poritz, K. Straub, P. J. Hoben, S. Brenner, and P. Walter, Nature (London), in press; K. Romisch, J. Webb, J. Herz, F. Prehn, R. Frank, M. Vingron, and B. Dobberstein, Nature (London) in press]. This protein, designated SRP54, contains a putative GTP-binding domain and a distinct, unusually methionine-rich domain. The properties of the latter domain led these investigators to suggest that it contains the signal sequence binding site. A previously uncharacterized 50-kDa E. coli protein, the sequence of which had been deduced from its gene sequence, was shown to exhibit homology in both domains. Similar GTP-binding domains were also found in the a- subunit of the SRP receptor (docking protein [section II B]) in the endoplasmic reticular membrane and in a second E. coli protein that resembles the SRP receptor. It was suggested that these GTP-binding proteins use GTP in sequential steps of the targeting reaction and that these essential features of the pathway are conserved from bacteria to mammals. Watanabe and Blobel (M. Watanabe and G. Blobel, Proc. Natl. Acad. Sci. USA 86: , 1989), purified an E. coli cytosolic factor to homogeneity and found it to be a homotetramer of SecB (64 kda with four identical 16-kDa subunits; section IV A). This homotetramer is claimed to be part of a larger 150-kDa complex that possesses the export factor activity (discussed in section IV C) which dissociates during purification. These workers suggest that the SecB tetramer functions in signal sequence recognition, as does the signal recognition particle of eucaryotes. Cheng et al. have recently identified a nuclear-encoded yeast mitochondrial heat shock protein, Hsp6O, that is essential for the assembly of matrix proteins into oligomeric complexes [M. Y. Cheng, F. U. Hartl, J. Martin, R. A. Pollack, F. Kalousek, W. Neupert, E. M. Hailberg, R. L. Hallberg, and A. L. Horwich, Nature (London) 337: , 1989]. Hsp6O was shown to be homologous to the E. coli GroEL protein and the Rubisco subunit-binding protein of chloroplasts discussed in section IV.A. Work in A. Kuhn's laboratory has led to the suggestion that an amphipathic a-helix at the N-terminus of the Pseudomonas aeruginosa Pf3 phage coat protein and the subse-

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