Membrane Potential. secretion of other secretory proteins is due to the mutant protein blocking these hypothetical export sites.

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1988, p /88/ $02.00/0 Copyright C) 1988, American Society for Microbiology Vol. 170, No. 5 Synthesis of an Escherichia coli Protein Carrying a Signal Peptide Mutation Causes Depolarization of the Cytoplasmic Membrane Potential N. STEPHEN POLLITT* AND MASAYORI INOUYE Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey Received 30 November 1987/Accepted 5 February 1988 A deletion mutation (IppA9Al3A14) in the signal peptide of the major outer membrane lipoprotein of Escherichia coli (Lpp) was found to cause severe effects on cell physiology, resulting in cessation of growth within 10 min of induction of lppa9al3al4 expression and rapid cell death. Further investigation revealed that lppa9al3al4 expression caused slow processing of several other exported proteins. The origin of this effect was traced to depolarization of the electrochemical potential across the cytoplasmic membrane, which is known to be required for efficient protein export. Analysis of the processing rate of the mutant, either prior to complete depolarization or in a suppressor strain in which depolarization does not occur, indicates that the mutant protein was capable of secretion at a rate which, while less than that of the wild type, was reasonably rapid compared with the rates of other E. coli secreted proteins. The existence of this type of signal peptide mutation suggests that the cell may have a mechanism to avoid membrane damage from secretory proteins carrying membrane-active signal peptides which is bypassed by the lppa9al3al4 mutant. In both procaryotic and eucaryotic species, the secretion of a protein across a biological membrane requires an amino-terminal extension, termed the signal peptide (for a review, see reference 17). Although they lack any strict sequence homology, signal peptides are generally characterized by a positively charged amino-terminal region followed by 8 to 15 hydrophobic residues ending in a less hydrophobic cleavage region. The conservation of these structural features suggests that they are important to signal peptide function, and several proposals have been made as to how these structures may function (9, 12, 22). Several studies have sought to test these ideas through the isolation and examination of export-defective proteins in which the structure of the signal peptide has been altered (3, 17). Although the effect of many of these mutations may be explained in terms of the extent to which they disrupt the structural motifs described above, many structurally conservative mutations have effects that cannot be explained based on the nature of the structural changes (17). In these cases, the underlying reason for the secretion defect induced by the signal peptide mutation is unclear. In addition to changes in secretion efficiency, signal peptide mutations can also cause changes in the level of expression of the secreted protein (11, 21), the general accumulation of secretory precursors within the cell (2, 14), as well as toxic effects on cell physiology (18, 20). These phenotypes hold some interest since they may provide clues as to the nature of the defect caused by the mutation. In particular, the accumulation of secretory precursors caused by a mutation or gene fusion altering the structure of only a single protein has been interpreted to suggest that a discrete number of export sites exist in the inner membrane and that the effect of a signal peptide mutation of one protein on the * Corresponding author secretion of other secretory proteins is due to the mutant protein blocking these hypothetical export sites. In the present study, we show that a prolipoprotein signal peptide mutant, lppa9a13a14, caused the accumulation of precursors to two other outer membrane proteins, LamB and OmpA. The cause of this effect is traced to depolarization of the electrochemical potential across the cytoplasmic membrane, which has been shown to be required for efficient protein secretion in vivo. In addition, the evidence suggests that membrane depolarization is not only responsible for the pleiotropic effect of the Lpp signal peptide mutations on the secretion of other outer membrane proteins but is also predominantly responsible for the poor secretion of the IppA&9A13AJ4 gene product itself. MATERIALS AND METHODS Bacterial strains and plasmids. Escherichia coli JA221 (hsdr reca leub6 lacy trpe5 F' laciq lac+ pro') or SB221 (JA221 lipp) was used as the host strain. Plasmid pa2b2p1-140 is a derivative of psp-a2b2,1 (18) and expresses IppA9AJ3AJ4 under the control of the lac operator. Pulse-labeling and immune precipitation. Cultures were grown in M9 medium (21) with 0.5% glycerol and 0.4% maltose as carbon sources. At a density of approximately 2 x 108/ml, expression in lpp was induced by the addition of 2 mm IPTG (isopropyl-p-d-thiogalactoside). Cultures were labeled by the addition of 50,uCi of [35S]methionine per ml. Labeling was terminated by the addition of ice-cold 10% trichloroacetic acid. The precipitated material from 1 ml of labeled culture was collected by centrifugation, dissolved in 1% (wt/vol) sodium dodecyl sulfate, and immunoprecipitated with antiserum to Lpp, OmpA, or LamB as previously described (18). Measurement of proline uptake rates. Rates of proline transport were measured essentially as previously described (7). [3H]proline (670,uCi, 67 Ci/mol) was added to 2 ml of

2 2052 POLLITT AND INOUYE mid-log-phase culture. At 10, 25, 40, and 55 s after proline addition, 0.3-ml samples were taken and filtered on a pum-pore-size nitrocellulose filter (BA85; Schleicher & Schuell, Inc.). Filters were immediately washed with 2 ml of M9 medium, dried, dissolved in 2-methoxyethanol, and subjected to scintillation counting. Selection of suppressor strains. Strain JA221 harboring plasmid pa2b2b1-140 was grown overnight in M9 liquid medium and then plated on LB plates (15) containing 2 mm IPTG. After overnight incubation at 37 C, colonies were picked atid checked for growth on M9 plates containing 0.2% Casamino Acids (Difco Laboratories) with or without 2 mm IPTG. Strains showing a high degree of IPTG resistance were cured of their plasmid and retransformed with pa2b2j Strains which remained IPTG resistant after retransformation were then checked for enhanced processing of lppa9aj3aj4 after 5 min of induction. RESULTS Previous characterization of IppA9AJ3AJ4 has indicated that this signal peptide mutant has two unusual properties (18). Induction of expression of the protein results in a cessation in cell growth within 10 min (18) and a greater than 10-fold reduction in viable count within 1 h (unpublished observation). Such a rapid effect on cell viability has not to our knowledge been previously observed in connection with a protein export defect. (It is important to note that although these observations were made by using expression from a multicopy vector, the expression level at full induction was lower than that of the strongly expressed chromosomal Ipp gene on a per copy basis. Staining of crude membrane fractions of strains expressing the wild-type gene from the chromosome and the fully induced multicopy vector show that the steady-state lipoprotein levels of the two strains are approximately equal.) Second, pulse-labeling experiments have indicated that while the precursor is 70% processed to the mature form, the remaining precursor is stable and apparently unavailable for processing (18). These two properties have caused us to investigate further the lppa9aj3aj4 phenotype. The observation that the IppA9AJ3AJ4 precursor did not undergo processing at a uniform rate suggests a mechanism in which the initial expression of the precursor might inhibit further export and processing. In such a case, we would expect to see the processing rate decrease over time after the induction of IppA9AJ3AJ4 expression. Processing of lppa9'a13a4 as a function of time after induction. Prolipoprotein mutant lppa9aj3aj4 is expressed from an inducible plasmid expression vector as previously described (18). Figure 1A shows a time course of Ipp A9A13A14 induction in an Alpp host strain (SB221) in which aliquots of the culture were pulse-labeled for 20 s with [35S]methionine at various times after induction and immunoprecipitated with antiserum to Lpp. Initially, processing of the IppA9AJ3AJ4 gene product was reasonably efficient, with greater than 50% processed to the mature form during the initial pulse-labeling. Shortly thereafter, however, accumulation of prolipoprotein became increasingly severe, until after 5 min of induction, only approximately 10% of the immunoprecipitated material was mature. General inhibition of protein export by lppa9al3al4 expression. Figure 1B depicts a pulse-labeling time course similar to that in Fig. 1A' except that for each time point, proteins OmpA, LamB, and Lpp were immunoprecipitated and the strain used in this experiment, JA221, expresses Lpp from both the wild-type and lppa9aj3aj4 mutant genes on the chromosome and plasmid, respectively. Since the wildtype Lpp (shown in the leftmost lane prior to induction of the mutant form) was rapidly processed, a blockage in processing of the wild-type Lpp may be inferred from the disappearance of mature Lpp in cells pulse-labeled after 5 min of lppa9a13aj4 induction (Fig. ib, third lane from left, lower panel). Although less severe than its effect on Lpp processing, the expression of lppa9aj3aj4 also had an effect on the rate of processing of both LamB and OmpA (Fig. 1B). In the case of LamB, the amount of mature form present after the 20-s pulse-labeling declined from 50 to approximately 20% of total LamB, while mature OmpA declined from 8Q to 40% based on densitometric scanning of the autoradiograms shown in Fig. 1B. The expression of all three proteins decreased over the course of the experiment, reflecting an overall decline in the rate of protein synthesis caused by 1ppA9AJ3A14 expression. Membrane depolarization by lppa9al3a14. The effect of lppa9aj3a14 expression on both export and protein synthesis suggests that expression of this signal peptide mutation may have an effect on the transmembrane electrochemical potential, which is important in protein export as well as in the energy metabolism of the cell (1, 6). To test this hypothesis, changes in the membrane potential during lppa9aj3aj4 induction were monitored by observing changes in the rate of proline uptake. Proline uptake is driven by the membrane potential and is frequently used as a qualitative mneasure of membrane potential fluctuations (6). A sharp decline in proline uptake ensued immediately after IppA9AJ3AJ4 induction, with uptake becoming virtually nil at 10 min after induction (Fig. 2). The rapid depolarization of the membrane indicated by this experiment could explain many of the acute effects of lppa9aj3aj4 expression on both cell growth and protein synthesis, particularly since ATP hydrolysis is a known consequence of membrane depolarization due to the action of the F1 ATPase (16). Selection for strains resistant to lppa9al3al4 expression. The acute lethality of lppa9aj3aj4 expression suggests the possibility of selection of suppressor strains no longer sensitive to its expression. Since IppA9A13AJ4 lethality is caused by an effect on the energy metabolism of the cell, a- p -.-. M -- i:-~f",! SB B. JA min M=-1 ḻ"4 LamB p --- " m ".. p J. BACTERIOL. OmpA =alpp FIG. 1. Accumulation of precursor during lppa9aj3a14 induction. Strains SB221 (AIpp) or JA221 (lpp+) harboring plasmid pa2b2,1-140 were induced for expression of IppA9AJ3AI4 by the addition of 2 mm IPTG. Pulse-labeling for 20 s was performed as described in Materials and Methods at the indicated times after induction. Labeled protein was analyzed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. P, Precursor; M, mature protein. For JA221, the antiserum used in immunoprecipitation is indicated on the right.

3 VOL. 170, 1988 SECRETION-INDUCED MEMBRANE DEPOLARIZATION 2053 w a w 0.4 -J_ TIME AFTER INDUCTION (min) FIG. 2. Membrane depolarization during IppA9A13A14 induction. Proline uptake rates were measured as described in Materials and Methods and plotted against time of induction of lppa9a13a14. Strains used were JA221 (0) and SP06 (0). strains resistant to IppA9A13A14 expression may contain mutations affecting maintenance of the transmembrane electrochemical potential as well as cell components important for Lpp export. Strains resistant to lppa9aj3aj4 expression were obtained by plating a culture of JA221 harboring plasmid pa2b2p1-140 on rich medium containing 2 mm IPTG. Resistant colonies appeared at a frequency of approximately Strains with plasmid-borne mutations were eliminated by curing the plasmid and retransforming with pa2b2pl Several strains which remained IPTG resistant after retransformation were examined for the ability to process IppA9AJ3AJ4 after 5 min. One of these, SP06, was chosen for further analysis. The effect of the suppressor mutation on 1ppA9A13A14- induced membrane depolarization is shown in Fig. 2 (closed circles). As expected based on the selection, the suppressor. strain was relatively resistant to lethal membrane depolarization caused by lppa9j13aj4 expression, with the rate of proline uptake declining less than 30%. Thus, by using the suppressor strain, we may be able to observe the efficiency of secretion of the 1ppA9AJ13J4 gene product without the complicating aspect of membrane depolarization. A time course of induction of lppa9a13aj4 in SP06 is shown in Fig. 3. Unlike the results obtained in strain JA221 (Fig. 1B), the processing of precursors for OmpA, LamB, or Lpp were no longer affected by the induction of lppa9a13a14 in the suppressor strain. Specificity of SP06 suppression. The ability of SP06 to suppress the effects of lppa9aj3aj4 expression on cell viability raises the question of its ability to suppress the lethal effects of other lpp signal peptide mutations. Figure 4 shows the results of spot tests for IPTG sensitivity carried out with lppa9aj3aj4 and three other lethal signal peptide mutations as well as mutations within the mature region which also cause lethal buildup of the Lpp precursor. The suppressor strain showed strong resistance to only one other mutation besides lppa9a13a14, i.e., to lppt20, a mutation at the signal peptide cleavage site (19). Although the lppt20 mutation is lethal, cell death occurs only after long-term expression of the mutant gene, apparently as a result of overaccumulation of the precursor protein in the membrane, and is not due to rapid membrane depolarization, as is the case with lppa9a13a14. Pulse-labeling of the suppressor strain expressing the lppt20 mutation showed no improve- SPO mii M- ' LamB t... OmpA M----i h Lpp FIG. 3. Suppression of precursor accumulation in SP06. Pulselabeling was performed after induction of lppa9aj3a14. The accumulation of precursors to LamB, OmpA, and Lpp was analyzed as described in the legend to Fig. 1. ment in the processing rate (data not shown), making it unlikely that suppression of the lethal phenotype of this mutation occurs via improvement of processing efficiency. DISCUSSION Although many mutants with defective signal peptides have been isolated, the underlying reason for the inability of the mutant signal peptide to function in the export of the protein has been determined for only a few. In the only case in which an extensive study was made, several mutations in the hydrophobic region of the LamB signal peptide were found to reduce the ability of the signal peptide to form an ct-helix (8). This is correlated with a reduced ability of the protein to insert into an artificial lipid bilayer (4). In the ctirrent study, we have demonstrated that the 1ppA9AJ3AJ4 precursor, previously found to be defective for I-pT21 DI L_ I IpA-i I 3A 1 4 Ip,pAlA.16:r pidi3t24- p A9r3 IC I 3 1T L I 2.z_ a SPO6 -IPTG +IPTG Inc easing Dilution FIG. 4. Allele specificity of suppressor activity. Stationaryphase cultures of either JA221 or SP06 were spotted onto minimal medium prepared with or without the addition of 2 mm IPTG. Successive 10-fold dilutions were also spotted to the left of the original culture as indicated. Alleles of lpp present on the plasmid expression vector are indicated on the left. Descriptions of these mutations are given in the following references: lppt20 and 1ppL20, reference 19; lppa9j3baj4, reference 18; IppAISA16, reference 20; IppI23I24 and lppa20i23124, reference 10; and 1(lpp-bla), reference 19. CF(Ipp-bla) is a fusion of the Lpp signal peptide plus nine amino acids of the mature region to P-lactamase.

4 2054 POLLITT AND INOUYE secretion (18), was in fact fully capable of reasonably rapid export from the cytoplasm. As a side effect of expression, however, depolarization of the transmembrane electrochemical potential occurred, disrupting the secretion of several outer membrane proteins, including that expressed by Ipp A9A13A14. Thus, the secretion defect is predominantly a secondary consequence of membrane depolarization. The reason for the observed membrane depolarization is not altogether clear. It should be pointed out, however, that depolarization caused by lppa9aj3aj4 expression occurred within minutes of induction and before an appreciable amount of the protein had accumulated in the cell. In contrast, several mutations of the hydrophobic region or the cleavage site of the Lpp signal peptide have deleterious effects on cell viability, including inability to form colonies on solid media and gradual growth inhibition after induction of synthesis in liquid media (19, 20). We believe this chronic lethal phenotype to be the result of general damage to the cytoplasmic membrane caused by the buildup of large amounts of unprocessed precursor protein. The much more rapid onset of lethality in the case of lppa9aj3aj4 makes it unlikely that such a mechanism operates here. In fact, after 5 min of lppa9aj3aj4 induction, the energy drain on the cell caused a general reduction in protein synthesis, to the effect that at no point did a significant amount of the lppa9aj3aj4 product accumulate. The ability of a small quantity of a secretory precursor to cause rapid cessation of growth and cell death points to the danger posed to the cell by the synthesis of secretory precursors with their associated membrane-active signal peptides. This suggests that the cell may in fact have a mechanism for avoiding such damage during the normal course of protein export. Interaction of the signal peptide with a cytoplasmic receptor might accomplish this function by shielding the signal peptide from the membrane prior to interaction with a membrane-bound receptor. Recent studies have, in fact, pointed to the existence of such a receptor (5). If this is the case, the lppa9aj3al4 precursor seems to have at least partially bypassed such a system. An alternative hypothesis is that, in fact, most signal peptides are finely tuned to avoid potentially harmful interactions with the cell membrane and that the lppa9aj3aj4 mutant has lost this ability. This seems unlikely, since, in fact, most signal peptides appear to be well suited for rapid interaction with the lipid bilayer, a property that has not only been verified experimentally in vitro but that also appears to be important to the ability of the signal peptide to promote export (4). The lppa9al3al4 allele is unlikely to have any greater membrane-active qualities, since hydrophobic residues have been deleted in this mutant. The mechanism of action of the suppressor mutation described here is unclear. Although it allowed expression of IppA9AJ3A14 without membrane depolarization, this effect was not achieved by suppression of the signal peptide defect, since in the only other case in which the lethal effects of a signal peptide mutation were suppressed by this mutation, the case of 1ppT2O, no improvement of processing was observed. Neither did the mutation cause a generalized resistance to membrane depolarization, since the strain remained resistant to proton ionophore carbonyl cyanide m-chlorophenylhydrazone. The ability of a single mutation to reverse both secretion and membrane depolarization illustrates the close connection between these two effects. ACKNOWLEDGMENTS We thank Donald Oliver for his generous gift of LamB antiserum and Tom Mammone for technical assistance. This work was supported by Public Health Service grant GM19043 (to M.I.) and fellowship GM10436 (to N.S.P.) from the National Institutes of Health. LITERATURE CITED J. BACTERIUOL. 1. Bakker, E. P., and L. L. Randall The requirement for energy during export of P-lactamase in Escherichia coli is fulfilled by the protonmotive force. EMBO J. 3: Bankaitis, V., and P. J. Bassford, Jr The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli. J. Biol. Chem. 259: Bankaitis, V. A., J. P. Ryan, B. A. Rasmussen, and P. J. Bassford, Jr The use of genetic techniques to analyze protein export in Escherichia coli. Curr. Top. Membr. Transp. 24: Briggs, M. S., L. M. Gierasch, A. Ziotnick, J. D. Lear, and W. F. DeGrado In vivo function and membrane binding properties are correlated for Escherichia coli LamB signal peptides. Science 228: Crooke, E., and W. Wickner Trigger factor: a soluble protein that folds pro-ompa into a membrane-assembly-competent form. Proc. Natl. Acad. Sci. USA 84: Date, T., J. M. Goodman, and W. R. Wickner Procoat, the precursor of M13 cost protein, requires an electrochemical potential for membrane insertion. Proc. Natl. Acad. Sci. USA 77: Date, T., C. Zwizinski, S. Ludmerer, and W. Wickner Mechanisms of membrane assembly: effects of energy poisons on the conversion of soluble M13 coliphage procoat to membrane-bound coat protein. Proc. Natl. Acad. Sci. USA 77: Emr, S. D., and T. J. Silhavy Importance of secondary structure in the signal sequence for protein secretion. Proc. Natl. Acad. Sci. USA 80: Engelman, D. M., and T. A. Steitz The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23: Ghrayeb, J., and M. Inouye Nine armino acid residues at the amino NH2-terminal of lipoprotein are sufficient for its modification, processing, and localization in the outer membrane of Escherichia coli. J. Biol. Chem. 259: Hall, M. N., J. Gabay, and M. Schwartz Evidence for a coupling of translation and export of an outer membrane protein in Escherichia coli. EMBO J. 2: Inouye, M., and S. Halegoua Secretion and membrane localization of proteins in Escherichia coli. Crit. Rev. Biochem. 7: Inouye, S., G. Duffaud, and M. Inouye Structural requirement at the cleavage site for efficient processing of the lipoprotein secretory precursor of Escherichia coli. J. Biol. Chem. 261: Ito, K., P. J. Bassford, Jr., and J. Beckwith Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell 24: Miller, J. H Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. Plate, C. 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5 VOL. 170, 1988 SECRETION-INDUCED MEMBRANE DEPOLARIZATION Pollitt, S., S. Inouye, and M. Inouye Effect of amino acid substitutions at the signal peptide cleavage site of the Escherichia coli major outer membrane lipoprotein. J. Biol. Chem. 261: Vlasuk, G. P., S. Inouye, and M. Inouye Serine and threonine residues within the signal peptide on the secretion of the major outer membrane lipoprotein of Escherichia coli. J. Biol. Chem. 259: Vlasuk, G. P., S. Inouye, H. Ito, K. Itakura, and M. Inouye Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J. Biol. Chem. 258: VonHeine, G., and C. Blomberg Transmembrane translocation of proteins. Eur. J. Biochem. 97: