The Sec translocon mediated protein transport in prokaryotes and eukaryotes

Transcription

1 Molecular Membrane Biology ISSN: (Print) (Online) Journal homepage: The Sec translocon mediated protein transport in prokaryotes and eukaryotes Kärt Denks, Andreas Vogt, Ilie Sachelaru, Narcis-Adrian Petriman, Renuka Kudva & Hans-Georg Koch To cite this article: Kärt Denks, Andreas Vogt, Ilie Sachelaru, Narcis-Adrian Petriman, Renuka Kudva & Hans-Georg Koch (2014) The Sec translocon mediated protein transport in prokaryotes and eukaryotes, Molecular Membrane Biology, 31:2-3, 58-84, DOI: / To link to this article: Published online: 24 Apr Submit your article to this journal Article views: 1506 View related articles View Crossmark data Citing articles: 49 View citing articles Full Terms & Conditions of access and use can be found at Download by: [Smithsonian Astrophysics Observatory] Date: 10 January 2018, At: 13:05

2 ISSN: (print), (electronic) Mol Membr Biol, 2014; 31(2 3): 58 84! 2014 Informa UK Ltd. DOI: / REVIEW ARTICLE The Sec translocon mediated protein transport in prokaryotes and eukaryotes Kärt Denks 1,2, Andreas Vogt 1,2,3, Ilie Sachelaru 1,2, Narcis-Adrian Petriman 1,2, Renuka Kudva 1,2,3, and Hans-Georg Koch 1,3 1 Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany, 2 Faculty of Biology, University Freiburg, Freiburg, Germany, and 3 Spemann Graduate School of Biology and Medicine (SGBM), Freiburg, Germany Abstract Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61a and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes. Introduction The sorting of proteins between different cellular compartments is mediated by a large diversity of protein transport systems (Lithgow & Waksman, 2013; Papanikou et al., 2007). In prokaryotes, the cytoplasmic membrane is responsible for asymmetric protein distribution between the cytosol and periplasmic space or the outer membrane in Gram-negative Bacteria. Targeting, sorting and transport systems in Eukaryotes are more complex, owing to the presence of organelles. The protein transport gateway to most organelles (secretory pathway) is the endoplasmic reticulum (ER) and the Sec translocon constitutes the major protein transport site in the ER membrane. The Sec translocon is also universally conserved in the cytoplasmic membrane of Bacteria and Archaea (Kudva et al., 2013; Zimmermann et al., 2011). In addition, the Sec translocon is present in the thylakoid membrane in chloroplasts, but absent in most mitochondria Correspondence: Hans-Georg Koch, Institut für Biochemie und Molekularbiologie, ZBMZ, Albert-Ludwigs-Universität Freiburg, Germany. Tel: / hans-georg.koch@ biochemie-uni-freiburg.de Kärt Denks, Institut für Biochemie und Molekularbiologie, ZBMZ, Albert-Ludwigs-Universität Freiburg, Germany. Tel: / kaert.denks@biochemie.uni-freiburg.de Keywords BiP, protein targeting, Sec61/SecYEG, SecA, SRP History Received 10 December 2013 Revised 27 February 2014 Accepted 10 March 2014 Published online 23 April 2014 with the exception of certain protozoans (Albiniak et al., 2012; Tong et al., 2011). Although the Sec translocon primarily functions as an aqueous conduit for proteins, it differs from most other pores as its channel forming subunit (SecY/Sec61a) is able to open both transversally into the periplasm/er lumen and also laterally to the lipid membrane (Figure 1). Transverse opening facilitates protein translocation across the membrane, whereas lateral opening of the translocon allows lipid insertion of membrane proteins. The lateral gate of the translocon is composed of four flexible a-helices of the SecY/Sec61a subunit (Van den Berg et al., 2004). The translocation of secretory proteins and large hydrophilic domains of membrane proteins through the SecY/Sec61 pore requires ATP hydrolysis by the ATPase SecA on the cis side of the bacterial membrane or by chaperones like BiP on the trans side of the ER membrane (Kudva et al., 2013; Zimmermann et al., 2011). The proton motive force (pmf) also contributes to the energetics of protein translocation across the bacterial membrane (van der Laan et al., 2004) although it is not essential for protein transport in vitro (Koch & Müller, 2000). A major challenge during protein transport is preventing premature and unproductive folding of proteins before they reach the Sec translocon. This is especially the case for hydrophobic membrane proteins which are 2014

3 DOI: / Eukaryotic and prokaryotic Sec translocon 59 Figure 1. Protein targeting pathways in bacterial and mammalian cells (A) Bacteria engage two targeting pathways for delivering proteins to the SecYEG translocon. The SecA-dependent pathway is used by periplasmic and outer membrane proteins, which contain a cleavable signal sequence. Cytosolic chaperones, including trigger factor and tetrameric SecB keep the nascent polypeptide in a translocation-competent state during its journey to the membrane. The pre-protein is then transferred to SecA, which drives translocation through the SecYEG channel by ATP hydrolysis. Although it is generally assumed that SecA acts post-translationally, some data indicate that SecA can bind to ribosome-nascent chains (RNCs), i.e. that it can also act co-translationally. The SecYEG translocon interacts at least transiently with the SecDFYajC complex, which might support the proton-motif force (pmf)-dependent steps during protein transport. The co-translational SRP-pathway is mainly used for inner membrane proteins and initiated by the ribosome-bound SRP. SRP-RNCs bind to the SecYEG-bound SRP receptor FtsY, RNCs dock onto the SecYEG translocon and the SRP-FtsY complex dissociates in a GTP-dependent manner. During the lateral exit from the SecYEG channel, the nascent membrane protein contactsyidc. YidC is shown to support SecYEG during membrane protein insertion, and it also acts as a SecYEG independent insertase for small or closely spaced membrane proteins. Targeting of membrane proteins to YidC also appears to be SRP-dependent. The translocon associates transiently with several additional proteins, that are required for cleaving the signal sequences of secretory proteins (signal peptidase, SPase), for protein folding (the periplasmic chaperones Skp and PpiD) or for quality control (the membrane-bound protease FtsH). (B) In eukaryotes, the Sec61-mediated insertion and the translocation occurs both co-translationally and post-translationally. During post-translational targeting, fully synthesized pre-proteins are kept in a transport-competent state by members of the Hsp90, Hsp70 and Hsp40 chaperone families. Translocation is mediated by the Sec61 complex in association with Sec62/Sec63 and the chaperone BiP at the lumenal side of the ER membrane. BiP binds to the translocating substrates in the ER lumen and prevents their back-sliding by an ATP-dependent ratcheting mechanism. The eukaryotic SRP pathway delivers both membrane proteins and secretory proteins co-translationally to the Sec61 complex. The eukaryotic SRP receptor consists of two unrelated GTPases, SRa (homologous to FtsY) and SRb. The Sec61 translocon associates in a substrate-dependent manner with additional proteins that either bind to RNCs (Ramp4) or are suggested to assist membrane protein folding (TRAM, Sec63). BiP is also required for co-translational transport. Like in bacteria, additional proteins are involved in processing and quality control (SPase; TRAP [translocon associated protein], oligosacharyl transferase [OST], the Hsp40-homologue Erj1 or AAAproteases). This Figure is reproduced in color in the online version of Molecular Membrane Biology.

4 60 K. Denks et al. Mol Membr Biol, 2014; 31(2 3): aggregation-prone. Targeting of many proteins therefore begins at the ribosome during translation. Co-translational targeting is initiated by the signal recognition particle (SRP), which binds to the tunnel exit of the ribosome to recognize its substrate early during synthesis (Berndt et al., 2009; Bornemann et al., 2008). SRP-ribosome-associated nascent chains (SRP-RNCs) are delivered to the membrane-bound SRP receptor (SR), and the ribosome thereafter aligns with the channel of the Sec translocon to facilitate co-translational transport of the nascent polypeptide (Gilmore et al., 1982b; Koch et al., 1999; Valent et al., 1998). In bacteria, the SRP pathway is mainly dedicated to the targeting of inner membrane proteins, while the eukaryotic SRP delivers both ER membrane proteins and secretory proteins to the Sec61 complex. The Sec translocon also transports proteins post-translationally, i.e. upon termination of protein synthesis. In bacteria, the post-translational mode is preferentially employed by periplasmic and outer membrane proteins and involves the cytosolic ATPase SecA (Koch et al., 1999; Koch & Müller, 2000). SecA also cooperates with the SRP pathway for the insertion of membrane proteins with periplasmic domains longer than approx. 30 amino acids (Deitermann et al., 2005; Neumann-Haefelin et al., 2000; Sääf et al., 2009). In eukaryotes, post-translational transport requires the association of Sec61 with the Sec62/Sec63 complex (Meyer et al., 2000; Panzner et al., 1995). The folding and processing of transported proteins is facilitated by periplasmic and lumenal chaperones (Gordon & Kindt, 1976; Missiakas et al., 1996), signal peptidases (Chang et al., 1978; Zwizinski & Wickner, 1980) and oligosaccharyl transferases (Lau et al., 1983). The final topology of membrane proteins is further affected by the lipid composition of the membrane (Dowhan & Bogdanov, 2009). The dynamic interplay of the core translocon with many additional factors is common to both prokaryotes and eukaryotes, and this probably ensures efficient transport and biogenesis of a vast array of substrates. This review provides an insight to the current knowledge on the Sec translocon. Most of the data on the prokaryotic Sec translocon is based on the Gram-negative model organism E. coli. For reviews covering protein translocation in Archaea and Gram positive bacteria please see Pohlschröder et al. (2005), Calo & Eichler (2011) and Yuan et al. (2010). Composition and architecture of the core Sec complex Most components of the Sec pathway were identified from genetic screens conducted in E. coli and Saccharomyces cerevisiae (Deshaies & Schekman, 1987; Emr et al., 1981; Oliver & Beckwith, 1981). Mutations that caused protein secretion defects were referred to as sec alleles and mapped to seca, secd, sece, secf and secy in E.coli; prl mutations (suppressors of signal sequence mutations) allowed the secretion of proteins with defective signal sequences and were mapped to seca (prld), sece (prlg), secg (prlh) and secy (prla) (Bieker & Silhavy, 1990; Emr et al., 1981, Ito et al., 1983; Oliver & Beckwith, 1981; Schatz & Beckwith, 1990). The three-dimensional crystal structure of the Sec translocon from the archaeon Methanocaldococcus janaschii confirmed many structural and functional predictions that were based on these early genetic screens (Smith et al., 2005; Van den Berg et al., 2004). Subsequent studies found that the general architecture observed for the Methanocaldococcus jananschii Sec complex appears to be universally conserved in both prokaryotes and eukaryotes (Becker et al., 2009; Clemons et al., 2004; Egea & Stroud, 2010; Frauenfeld et al., 2011; Gogala et al., 2014; Gumbart et al., 2009; Li et al., 2007; Ménétret et al., 2005; 2007; 2008; Mitra et al., 2005; Park et al., 2014; Tsukazaki et al., 2008; Zimmer et al., 2008). The core Sec translocon consists of three protein subunits SecY, SecE and SecG in bacteria, and Sec61a, Sec61g and Sec61b in eukaryotes (Zimmermann et al., 2011). SecYE and Sec61ag exhibit significant sequence conservation and are essential (Kudva et al., 2013; Park & Rapoport, 2012). The Sec61b subunit found in Eukaryotes and Archaea is not homologous to the eubacterial SecG subunit, and neither Sec61b nor SecG are essential for protein transport (Finke et al., 1996; Nishiyama et al., 1994). SecY and Sec61a are comprised of 10 transmembrane a-helical domains (TMs) each and have similar molecular masses, i.e. 48 kda for E. coli SecY and 52 kda for Homo sapiens Sec61a (Rensing & Maier, 1994). When visualized from the top, the 10 TMs are divided into two halves that resemble a clamshell surrounding a central pore (Figure 2D; Van den Berg et al., 2004). The two halves (TMs 1 5 and 6 10) are connected by a periplasmic loop, which is referred to as the hinge region or the back of the translocon. A side section through the SecYEb complex reveals an hourglassshaped structure with two funnels, one opening to the cytoplasmic face and the other one to the periplasmic face of the membrane (Figure 2A). The two funnels are separated by a central constriction, which is called the pore ring. It is comprised of amino acid residues with bulky hydrophobic side chains, e.g. by six isoleucine residues in E. coli SecY (Van den Berg et al., 2004). The translocon assumes a closed conformation in the resting state: the cytoplasmic funnel is empty and its periplasmic counterpart is plugged by a short a-helix (Helix 2a or plug) (Tsukazaki et al., 2008; Van den Berg et al., 2004). Mutagenesis studies and structural data suggest that the central constriction and the plug may play a role in the controlled opening and closing of the Sec pore (Egea & Stroud, 2010; Harris & Silhavy, 1999; Van den Berg et al., 2004). Molecular dynamics simulations support the view that the plug participates in sealing the pore and maintaining substrate selectivity of the translocon (Gumbart & Schulten, 2008). However, deleting the plug domain of the Sec channel does not affect growth of either E. coli or S. cerevisiae (Junne et al., 2006; Li et al., 2007; Maillard et al., 2007) although it decreases the selectivity of the translocon for its substrates (Li et al., 2007; Maillard et al., 2007). The X-ray structure of the plug-less SecYE channel shows that neighbouring residues can replace the function of the plug (Li et al., 2007). The exit of TMs into the lipid phase is facilitated by structural rearrangements in the lateral gate of the SecY/ Sec61a channel and involves TMs 2b and 3 on one side

5 DOI: / Eukaryotic and prokaryotic Sec translocon 61 Figure 2. The Sec translocon. Schematic representation of the Sec translocon in the closed (A) and the open (B) conformation viewed from the front in the membrane plane (left), as a transverse section through the middle of the pore in the membrane plane (middle) and from the cytoplasmic side (top, right). The open and the closed conformation refer to the lateral gate being closed or open as shown in the front and top representation, respectively. The transverse section and the top view show the pore ring and the plug being displaced for the accommodation of a substrate. (C) Surface representation of the Archaeal SecYEb translocon in the plane of the membrane (adapted from Van den Berg et al. [2004]; pdb: 1RHZ). The lateral gate helices (TM2b, TM3, TM7 and TM8) of SecY and a short helix (helix 2a), called the plug, are highlighted. The plug is suggested to be involved in sealing the channel. The cytoplasmic loops C4, C5 and C6 of SecY are the major cytoplasmic contact sites for FtsY, SecA and the ribosome. (D) The top view of Sec61YEb from the cytoplasmic site shows the plug (dark green) sealing the channel and SecE embracing SecY at the back. This Figure is reproduced in color in the online version of Molecular Membrane Biology. and helices 7 and 8 on the other side of SecY (Hizlan et al. (2012), Figure 2C). Signal sequences might be maintained at the lateral gate since cross-linking data have shown that signal sequences contact lipids during insertion (Higy et al., 2005; Martoglio et al., 1995) and that they are intercalated between transmembrane helices 2 and 7 of SecY/Sec61a (Plath et al., 1998). Most bacteria have a SecE molecule with only one TM. In contrast, E. coli SecE consists of three TMs and has a molecular mass of 14 kda. However, only the third TM of SecE is essential for protein transport and cell viability (Schatz et al., 1991). In eukaryotes, including H. sapiens, Sec61g is a single-spanning membrane protein of approx. 8 kda (Hartmann et al., 1994). SecE is located at the back of SecY (Figure 2D) stabilizing the two halves of SecY (Van den Berg et al., 2004). Indeed, SecY molecules that have been proteolytically cleaved at the hinge region remain active if SecE is present (Lycklama a Nijeholt et al., 2013). SecY in E. coli is rapidly degraded by the membrane protease FtsH in the absence of SecE (Kihara et al., 1995). SecG in E. coli is a 12 kda protein consisting of two TMs connected by a cytoplasmic loop. Cross-linking studies have located SecG next to the cytosolic loops C2 and C3 of SecY (Satoh et al., 2003; van der Sluis et al., 2002), although SecG/ Sec61b are thought to have only limited contacts to SecY and SecE (Van den Berg et al., 2004; Zimmer et al., 2008). SecG is not essential for protein transport in vitro (Brundage et al., 1990) but SecG deletion strains exhibit protein transport defects in vivo (Flower, 2001; Flower et al., 2000). In E. coli, the function of SecG has been linked to the SecA-dependent post-translational transport across the Sec channel (Duong & Wickner, 1997b; Morita et al., 2012; Nishiyama et al., 1996). As SecA is absent in Eukaryotes with the exception of chloroplasts and since it is also not found in Archaea, a functional connection between SecA and SecG would explain its presence in Bacteria only. SecG has been proposed to undergo reversible topology inversions for facilitating SecA- SecY interaction (Morita et al., 2012; Nishiyama et al., 2012; Sugai et al., 2007). Although several dual topology proteins have been identified in E. coli (Daley et al., 2005; Rapp et al., 2006), a topologically fixed SecG derivative does not prevent SecA-dependent protein translocation (van der Sluis et al., 2006). Thus, the physiological significance of the topology switch needs to be further analysed. Recent data suggest that the orientation of SecG depends on a non-proteinaceous glycolipozyme that was shown to influence membrane protein insertion and translocation (Moser et al., 2013). The non-homologous Sec61b in Eukaryotes and Archaea is slightly smaller than SecG and contains only one TM (Hartmann et al., 1994; Kalies et al., 1998). Sec61b was shown to interact with the SRP receptor (Helmers et al., 2003) and with the signal peptidase in yeast (Kalies et al., 1998). Furthermore, the role of Sec61b might not be limited to translocation since it interacts with Rtn1p, a protein involved in ER tubule formation (Zhao & Jäntti, 2009), and it appears to be required for plasma membrane targeting of Gurken, the ligand of epidermal growth factor receptor in Drosophila (Kelkar & Dobberstein, 2009). Additional subunits, partner proteins and the membrane environment of the bacterial and eukaryotic Sec complex The Sec complex has a modular nature. Some of the interactions of the core Sec translocon have been observed in all domains of life, e.g. with ribosomes, the SRP receptor

6 62 K. Denks et al. Mol Membr Biol, 2014; 31(2 3): Table 1. Sec-translocon associated proteins and their conservation. Dark grey represents the proteins present in all or most species; light grey represents the proteins found in some species; blank no known homologue. The paralogues are not indicated. and signal peptidases; yet many interactions are characteristic to either Bacteria or Eukaryotes. A number of proteins have been shown to interact at least transiently with the Sec translocon (Table 1). Attempts to determine the structure of a holo-translocon (Duong & Wickner, 1997b) comprising several modules attached to the Sec core complex have been successful for SecA-SecYE complexes (Zimmer et al., 2008) and for ribosome-secyeg/ribosome-sec61 complexes (Frauenfeld et al., 2011; Gogala et al., 2014; Ménétret et al., 2005, 2007, 2008; Mitra et al., 2005; Park & Rapoport, 2012; Park et al., 2014). The Sec translocon interactions with additional partners depend on the nature of the substrate and therefore the exact composition of the holo-translocon is probably rather flexible in vivo. The interaction of ribosomes with Sec complex The ability of the Sec complex to bind to ribosomes is an essential feature and ribosome binding sites on the translocon are evolutionarily conserved (Becker et al., 2009; Frauenfeld et al., 2011; Houben et al., 2005; Prinz et al., 2000). The cytosolic loops of SecY/Sec61a between TM 6 and 7 (C4 loop) and TM 8 and 9 (C5 loop) mediate ribosome binding (Cheng et al., 2005; Frauenfeld et al., 2011; Kuhn et al., 2011; 2014; Park & Rapoport, 2012; Park et al., 2014; Yeast Mammals Bacteria Archaea Mitochondria Chloroplasts References Translocon SecY/Sec61a * SecE/Sec61g * SecG Sec61b Translocon-associated proteins SecD/SecF YajC YidC/Oxa1/Alb3 Sec62 Sec63 Sec71/72 ERj1 TRAM TRAP Ramp4/Ysy6p SecA BiP/Kar2 Calmodulin Targeting factors SRP SR Processing enzymes Spase1 OST FtsH** Chaperones SecB Skp PpiD 1 Hartmann et al. (1994); 2 Cao & Saier (2003); 3 Pohlschröder et al. (2005); 4 Cline & Dabney-Smith (2008); 5 Kinch et al. (2002); 6 Eichler (2003); 7 Hand et al. (2006); 8 Tseng et al. (1999); 9 Zhang et al. (2009b); 10 Tyedmers et al. (2000); 11 Dudek et al. (2002); 12 Yamaguchi et al. (1999); 13 Nohara et al. (1995); 14 Nakashima et al. (2012); 15 Dalbey et al. (1997); 16 Calo & Eichler (2011); 17 Nothaft & Szymanski (2010); 18 Aebi et al. (2013); 19 Janska et al. (2013); 20 van der Sluis & Driessen (2006); 21 Gatsos et al. (2008). *SecYE is only found in mitochondria of some protozoans (Albiniak et al., 2012; Tong et al., 2011); **Eukaryotes have other members of AAA-metalloproteases. 1,2,3,4 1,2,3,4 1,2,3,4 1,2,3,5 6,7,8 Raden et al., 2000). The universal ribosome adaptor site consisting of the proteins L23, L24 and L29 (E. coli nomenclature, Figure 3), and conserved rrna helices, establish contacts to the Sec translocon both in prokaryotes and eukaryotes (Becker et al., 2009; Frauenfeld et al., 2011). Recent comparative cryo-em reconstructions show that both translating and non-translating ribosomes provide the same binding sites for the translocon although rather large conformational changes take place within the translocon upon substrate binding (Gogala et al., 2014; Park et al., 2014). The tunnel exit area of the ribosome contacts not only the Sec translocon, but acts as the binding platform for SRP, SecA, trigger factor, and nascent chain modifying enzymes (Figure 3), (Baram et al., 2005; Ferbitz et al., 2004; Frauenfeld et al., 2011; Gu et al., 2003; Huber et al., 2011; Kramer et al., 2002; Kramer et al., 2009; Kuhn et al., 2011; Schlünzen et al., 2005; Ullers et al., 2003). Similarly, the corresponding area of the eukaryotic ribosome also controls the binding of SRP, methionine aminopeptidase 1 and the chaperones NAC (nascent chain associated complex) and Ssb1/2 in a substrate-specific manner (Raue et al., 2007). It is not clear how binding of that many ribosomal tunnel exit ligands is coordinated in space and time. The interaction of other ribosomal regions with the membrane might also facilitate the contact to the Sec translocon. 2,6 9 3,10 3, , ,3 2, ,17,

7 DOI: / Eukaryotic and prokaryotic Sec translocon 63 Figure 3. The ribosomal tunnel exit as a binding platform for targeting factors, chaperones, nascent chain processing enzymes and the translocon. L23, L24 and L29 constitute a universal ribosomal adaptor site. Data are collected from: TF and peptidyl formylase (PDF): (Kramer et al., 2002; Bingel-Erlenmeyer et al., 2008); SecY: (Frauenfeld et al., 2011); SecA: (Huber et al., 2011); SRP: (Gu et al., 2003; Halic et al., 2006; Schaffitzel et al., 2006); methionine amino peptidase (MAP): (Sandikci et al., 2013); YidC: (Köhler et al., 2009; Seitl et al., 2013; Welte et al., 2012). This Figure is reproduced in color in the online version of Molecular Membrane Biology. One such region is the eukaryotic ribosome expansion segment 27 (ES27 L ) which has been shown to contact the ER membrane by in situ cryo-em tomography in canine pancreas microsomes (Pfeffer et al., 2012). Conformational rearrangements of ES27 L might play a role in ribosome release from the ER membrane (Pfeffer et al., 2012). Whether ES27 L interacts with the membrane lipids or proteins is not yet clear but its flexibility suggests that it might respond to events on the ribosomal tunnel exit. The interaction of the SRP receptor with Sec complex The transfer of RNCs from the SRP to the Sec complex is the final and most crucial step during co-translational targeting. Biochemical and genetic evidence suggest that membranebound SR interacts directly with the Sec61 complex (Jiang et al., 2008; Song et al., 2000). The bacterial SR is termed FtsY and it is homologous to the eukaryotic SRa subunit (Luirink et al., 1994). FtsY and SRa belong to the SIMIBI family of GTPases harboring a characteristic NG-domain (Figure 4C). However, both proteins use different strategies for membrane binding. The X-domain of SRa dimerizes with SRb, an integral 30 kda ER membrane protein present only in Eukaryotes (Figure 4E) (Schwartz & Blobel, 2003; Schlenker et al., 2006). SRb belongs to the Ras superfamily of small GTPases and it requires GTP-activation to allow stable SRa binding (Fulga et al., 2001; Schwartz & Blobel, 2003). The observation that Sec61 regulates the nucleotide occupancy of SRb has led to the idea that Sec61b functions as nucleotide exchange factor for SRb (Helmers et al., 2003). The interaction with Sec61b could keep SRb in its GTPbound state, which would prime it for the subsequent interaction with SRa. There is no SRb-homologue present in bacterial membranes and as FtsY does not have an X-domain, it uses both its NG domain and an enterobacteria-specific A-domain for membrane attachment. FtsY binds to negatively charged phospholipids and to the cytosolic loops of SecY (Angelini et al., 2005; 2006; Braig et al., 2009; Kuhn et al., 2011; Parlitz et al., 2007). In E. coli, FtsY is present in large excess over SecYEG and it is likely that a large portion of the SecYEG translocons are in contact with FtsY (Drew et al., 2003; Kudva et al., 2013). Importantly, the same conserved residues of SecY that are in contact with FtsY also bind to the ribosome (Kuhn et al., 2011). Therefore, it appears likely that FtsY occupies the ribosome binding site of SecY until it is displaced by SRP-RNCs. FtsY also competes with SecA for SecYEG binding (Kuhn P, Koch HG, unpublished work), but it is unknown how access of FtsY or SecA to SecYEG is regulated in vivo. Although the A-domain of FtsY has been shown to interact with SecY, deleting the A-domain reduces the efficiency of co-translational targeting only moderately (Eitan & Bibi, 2004; Weiche et al., 2008). In contrast, deleting the two lipid-binding helices in the N-terminus and at the interface of A and N domains of FtsY completely inhibits co-translational targeting (Parlitz et al., 2007; Weiche et al., 2008). This could indicate that membrane attachment of FtsY and not its ability to bind to SecY is crucial for its function (Mircheva et al., 2009). However, a second SecY binding site is proposed to exist within the NG-domain of FtsY, which could facilitate its binding to SecY independently of the A-domain (Kuhn et al., 2011). In addition, the membrane around the bacterial Sec complex is likely enriched with phosphatidylglycerol and cardiolipin, which are required for SecYEG activity (Gold et al., 2010). As FtsY also binds preferentially to negative phospholipids (Braig et al., 2009), sufficient amounts of FtsY are probably located in close proximity to the SecYEG complex even in the absence of the A-domain. The interaction of signal peptidase with Sec complex The majority of non-cytosolic proteins originally bear a signal sequence which is recognized by targeting factors. After the pre-protein is targeted to the translocase, the signal sequence is eventually removed by membrane-embedded signal peptidases (SPases). The signal peptide is subsequently degraded by the membrane bound signal peptide peptidases (SPPase) (Nam & Paetzel, 2013; Voss et al., 2013; Wang et al., 2008) and the amino acids are recycled. Many different SPases are found in all domains of life. Prokaryotic SPases are classified as Spase I, II and IV (Auclair et al., 2012). The Spase I (LepB in E. coli) is an essential and conserved serine-protease, specific to the non-lipoprotein substrates of the Sec and Tat (twinarginine-dependent translocation) translocons (Auclair et al., 2012; Nyathi et al., 2013). Its catalytic domain is located in the periplasm and its TMs probably assist in signal peptide processing (Paetzel et al., 1998). The Spase I of E. coli has

8 64 K. Denks et al. Mol Membr Biol, 2014; 31(2 3): Figure 4. Structure of the signal recognition particle (SRP) and its receptor (SR) (A) Cryo-EM reconstitution of the eukaryotic SRP with the conserved SRP54 subunit, the additional eukaryotic SRP subunits and the 7.5 S RNA (adapted from: (Halic et al., 2004); pdb: 1RY1). (B) Crystal structure of the prokaryotic SRP (adapted from (Ataide et al., 2011); pdb: 2XXA). The conserved protein subunit Ffh (fifty-four homologue) and the 4.5 S RNA. (C) Crystal structure of the NG-subunit of the bacterial SRP receptor FtsY (adapted from (Ataide et al., 2011); pdb: 2XXA) (D) Complex of the prokaryotic SRP and the NG-domain of FtsY (adapted from (Ataide et al., 2011); pdb: 2XXA) (E) Crystal structure of the eukaryotic X-domain of SRa in complex with the cytoplasmic domain of SRb (adapted from Schwartz & Blobel [2003]; pdb: 1NRJ). This Figure is reproduced in color in the online version of Molecular Membrane Biology. two transmembrane domains while Bacillus subtilis has one (Tjalsma et al., 1998). Eukaryotic signal peptidases are organized in multi-subunit complexes termed SPC. However, the catalytic activity of SPC is located at LepB homologue which is Sec11 in yeast (Figure 5) (VanValkenburgh et al., 1999) and Spc18/Spc21 in mammals (Liang et al., 2003). Although signal sequences of Sec substrates are cleaved off during the translocation (Josefsson & Randall, 1981a; 1981b), evidence for a direct interaction between SPase and the translocon is limited. So far, only the yeast Sec61b was shown to interact with signal peptidase during translocation (Antonin et al., 2000; Kalies et al., 1998). The effect of the lipid environment on protein transport The lipid bilayer constitutes the permeability barrier of the cell and influences directly the activity of multiple membrane protein complexes, including the Sec translocon. Phospholipids also affect the stability and final topology of newly synthesized membrane proteins (Dowhan & Bogdanov, 2009). This is achieved by the charged phospholipid head groups, their asymmetric distribution and the nature of the acyl chains. The ER membrane and the cytoplasmic membrane of Gram-negative bacteria consist largely of zwitterionic phospholipids while Gram-positive bacteria have more anionic membrane lipids (Epand & Epand, 2011). A comparison of the lipid composition of E. coli membrane and the ER membrane is given in Table 2. The zwitterionic phospholipid phosphotidylethanolamine (PE) is important for membrane elasticity and curvature (Raetz, 1978). PE probably supports conformational flexibility of the Sec complex during protein transport (Rietveld et al., 1995) and its depletion has been shown to reduce translocation efficiency (Mikhaleva et al., 2001; van der Does et al., 2000). PE has also been suggested to affect membrane binding of FtsY (Millman et al., 2001). Phospholipids with negatively charged head groups like phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphatidylinositol (PI) have the most pronounced effect on membrane protein biogenesis (Table 3). The activities of both SecA and FtsY are stimulated by negatively charged

9 DOI: / Eukaryotic and prokaryotic Sec translocon 65 Figure 5. The topology of yeast Sec62, Sec63, Sec71, Sec72 and Sec11. The lumenal J-domain of Sec63 is important for the recruitment of BiP, a chaperone which is essential for Sec61-mediated translocation. The negatively charged C-terminus of Sec63 binds to the N-terminus of Sec62 to collectively support post-translational translocation. Sec71 and Sec72 form the complex with Sec62/Sec63. Sec11 is the catalytic subunit of the yeast signal peptidase complex. This Figure is reproduced in color in the online version of Molecular Membrane Biology. Table 2. The phospholipid composition of the E. coli inner membrane and the ER membrane. Structural lipids of the E. coli inner membrane 1 Structural lipids in ER membrane 2,3 phospholipids (Bahari et al., 2007; Braig et al., 2009; Lam et al., 2010; Lill et al., 1990; Parlitz et al., 2007) and the absence of PG severely impairs protein transport in E. coli (de Vrije et al., 1988; van der Does et al., 2000). Cardiolipin (CL) also stabilizes the SecYEG dimer and creates a high affinity binding surface for the motor protein SecA (Gold et al., 2010). Acidic phospholipids induce the dissociation of dimeric SecA exposing its binding interface to SecYEG (Alami et al., 2007). The amount of CL bound to SecYEG is proportional to the ATPase activity of SecA (Gold et al., 2010). Sterols seem to inhibit protein translocation initiation, most likely hindering RNC binding to Sec61 (Nilsson et al., 2001; Yamamoto et al., 2012). This is probably the reason why sterols are scarce in the ER membrane (van Meer et al., 2008). The interaction network of bacterial Sec complex PE PG CL PC PE PS PI Charge zwitterionic anionic anionic zwitterionic zwitterionic anionic anionic Coverage 70 75% 20 25% 5 10% 50 60% 25 30% 1 5% 10 15% Bilayer formation* PE, phosphatidylethanolamine; PG, Phosphatidylglycerol; CL, Cardiolipin; PC, Phosphatidylcholine; PS, Phosphatidylserine; PI, Phosphatidylinositol. *Bilayer formation designates the ability to spontaneously organize into a lipid bilayer in the correct solvent. 1 van der Does et al. (2000); 2 Raetz (1978); 3 van Meer et al. (2008). Table 3. Influence of structural lipids of the E. coli inner membrane and the ER membrane on targeting and function of Sec translocon. Structural lipids of E. coli inner membrane Structural lipids of ER membrane PE PG CL PC PE PS Targeting ,5 n.a Insertion ,5 ++ 3,5 n.a n.a. Translocation , Folding ++ 1,2,8, ,5 n.a. ++ important; + important but incompletely investigated; n.a., no information available. 1 Bogdanov et al. (1996); 2 Vitrac et al. (2011); 3 van Dalen & de Kruijff (2004); 4 Gold et al. (2010); 5 Braig et al. (2011); 6 Kusters et al. (1991); 7 Yamamoto et al. (2013); 8 Bogdanov & Dowhan (2012); 9 Bogdanov & Dowhan (1998); 10 Dowhan & Bogdanov (2009). Some Sec complex-associated proteins like SecA are present only in bacteria while others like periplasmic chaperones also have functional homologues in eukaryotic cells (Table 1). SecA SecA is (Figure 6) probably best studied partner protein of the bacterial Sec complex. It was identified in the genetic screens during the discovery of the sec and prl alleles (Emr et al., 1981; Oliver & Beckwith, 1981). It has a dual role as it acts as an ATP-fueled motor supporting protein transport across the inner membrane and as a targeting factor for the posttranslational pathway. SecA binds to SecYEG with an affinity of nm (Douville et al., 1995) and is considered to function as a soluble subunit of the Sec translocon. SecA and FtsY have an overlapping binding sites on SecY, however SecA binds to additional residues on SecY that are distributed across cytosolic loops C2 C6 (Mori & Ito, 2006). The crystal structure of SecA in complex with the bacterial translocon (Zimmer et al., 2008) shows SecA:SecYEG in a 1:1 stoichometry but biochemical data also support a 1:2 or 2:2 stoichometry (Deville et al., 2011; Osborne & Rapoport, 2007). The PBD domain (peptide binding domain); also called PPXD; (pre-protein cross-linking domain) of SecA, provides the major contact site with SecY (Figure 6C). The PBD domain of free SecA is closely packed against the helical wing domain (HWD) (Hunt et al., 2002; Vassylyev et al., 2006) but in the SecY-bound SecA structure the PBD is rotated towards the nucleotide-binding domain 2 (NBD-2) (Zimmer et al., 2008). It is thought that the PBD domain functions as a flexible trap that captures the translocating substrate in a groove formed by PBD and HWD (Park & Rapoport, 2012). In the SecA-SecYE structure, the groove is aligned with the SecY channel, allowing the substrate to move through the groove into the channel (Zimmer et al., 2008).

10 66 K. Denks et al. Mol Membr Biol, 2014; 31(2 3): Figure 6. Structure of SecA, the motor protein of the post-translational transport in bacteria. (A) Schematic domain organisation of SecA (NBD, Nucleotide binding domains; PBD, peptide-cross-linking domain; HSD, helical scaffold domain; HWD, helical wing domain; CTD, C-terminal domain). (B) Crystal structure of SecA from Thermotoga maritima (adapted from Zimmer et al. (2008); pdb: 3DIN). The colour code is the same as in (A). (C) Crystal structure of SecA in complex with the SecYEG translocon (adapted from Zimmer et al. (2008); pdb: 3DIN). The helices of the lateral gate of SecY are highlighted. This Figure is reproduced in color in the online version of Molecular Membrane Biology. SecDFYajC The trimeric SecDFYajC complex is a low-abundant integral membrane protein complex that was shown to interact with the SecYEG (Duong & Wickner, 1997b). The deletion of SecD/SecF negatively affects bacterial growth and their presence stimulates protein export (Pogliano & Beckwith, 1994). SecDFYajC might support the pmf-and SecA-dependent steps of protein transport (Duong & Wickner, 1997a; Tsukazaki et al., 2011). However, Archaea lack SecA but have SecDF, thus their SecA-associated role is not clear (Hand et al., 2006). As SecDFYajC binds to the YidC insertase, it was proposed to tether YidC to the SecYEG channel (Nouwen & Driessen, 2002). However, a recent study showed that SecY and YidC interact even in the absence of SecDF (Sachelaru et al., 2013). YidC YidC is an essential membrane protein, present in Bacteria, some Archaea, mitochondria (Oxa1) and chloroplasts (Alb3, Alb4) (for review see Dalbey et al., 2011; Kudva et al., 2013). It acts as a co-insertase/chaperone supporting the integration of membrane proteins via the Sec complex (Beck et al., 2001; Nagamori et al., 2004). YidC was recently shown to establish extensive contacts to all four TMs of the lateral gate of SecY (Sachelaru et al., 2013). Apart from that, YidC can also serve as a Sec-independent insertase for a broad range of inner membrane proteins (Chen et al., 2002; Samuelson et al., 2001; Welte et al., 2012). YidC substrates are mainly hydrophobic without long periplasmic stretches (Welte et al., 2012). While targeting of substrates to YidC has been shown to require the SRP pathway (Facey et al., 2007; Welte et al., 2012), it remains to be investigated whether this is a general rule for targeting. It also remains to be studied how YidC-mediated insertion of membrane proteins occurs in vivo. PpiD and Skp The periplasm of Gram-negative bacteria hosts a myriad of chaperones engaged in protein folding and quality control (for review see Merdanovic et al., 2011). Two of these periplasmic chaperones, PpiD and Skp (seventeen-kilodaltonprotein) are known to act in the immediate vicinity of the SecYEG translocon. PpiD and Skp are periplasmic chaperones that influence the assembly of numerous outer membrane and periplasmic proteins (Chen & Henning, 1996; Dartigalongue & Raina, 1998; Jarchow et al., 2008). Skp was shown to interact with its substrate in the vicinity of the plasma membrane (Schäfer et al., 1999) and before the preprotein is fully translocated by the Sec complex (Harms et al., 2001). Although this suggests that Skp is in close proximity to the Sec complex, direct evidence for an interaction between the two is lacking. This is different for PpiD, another non-essential and membrane-anchored periplasmic chaperone. Cross-linking data show that PpiD establishes extensive contacts with the lateral gate of SecY (Sachelaru et al., 2013). PpiD is thought to mediate the release of the nascent chain from the translocon and it could play a role in the early folding of translocated proteins (Antonoaea et al., 2008; Matern et al., 2010). FtsH and Syd FtsH is an essential zinc-metalloprotease which plays a role in membrane protein quality control in bacteria, mitochondria and chloroplasts. It is proposed to degrade misfolded substrates in an ATP-dependent fashion (Dalbey et al., 2012; Ito & Akiyama, 2005). FtsH has been shown to degrade the SecY subunit of the translocon when SecE is not present in stoichiometric amounts (Kihara et al., 1995). This could be mediated by the small SecY-binding cytosolic protein Syd which might recognize the compromised status of the translocon (Dalal et al., 2009). FtsH has also been found in the complex with YidC indicating that the latter might participate in the quality control of transport processes (van Bloois et al., 2008). MPiase MPiase is a glycolipid composed of diacylglycerol and a glycan chain of three acetylated aminosugars linked through

11 DOI: / Eukaryotic and prokaryotic Sec translocon 67 pyrophosphate. MPiase was shown to exhibit chaperone-like activity driving subsequent membrane integration of substrates (Nishiyama et al., 2012). A role during protein translocation and a direct interaction with the Sec complex has also been suggested based on the observation that the topology inversion of SecG occurs only when MPiase associates with SecYEG (Moser et al., 2013). The interaction network of eukaryotic Sec complex The core Sec complexes in eukaryotes have additional subunits like Sec62 and Sec63, which are involved in posttranslational protein transport. The intricate interaction network of the Sec61 translocon also includes proteins required for energizing protein transport (BiP) and substrate folding and modification (TRAP, TRAM, OST). A recent study identified also O-mannosyltransferase in complex with the Sec translocon and found that mannosylation can take place during translocation of the substrate protein (Loibl et al., 2014). The eukaryotic translocon also establishes transient contacts to protein kinases and protein acetylases since Sec61 is a subject of co-translational and post-translational regulation. Sec61b, ERj1 and Sec63 have been shown to be phosphorylated by protein kinase C and casein kinase 2 (Ampofo et al., 2013; Götz et al., 2009; Gruss et al., 1999). Yeast Sec62 and Sec61b (Sbh1) appear to be co-translationally acetylated by NatA (Soromani et al., 2012). Sec62/Sec63 Sec62 and Sec63 are integral ER membrane proteins facilitating Sec61-dependent translocation. Sec62 has two membrane-spanning helices and a positively charged N-terminal cytoplasmic domain (Wittke et al., 2000). Sec63 belongs to the Hsp40 family of heat shock proteins and contains a characteristic J-domain between its second and third TM (Figure 5) (Skowronek et al., 1999). The J-domain is located in the ER lumen where it interacts with the Hsp70- family protein BiP (Brodsky et al., 1995). The negatively charged C-terminus of Sec63 contacts the N-terminus of Sec62 (Lang et al., 2012). Sec62 is an essential protein involved in posttranslational translocation (Deshaies & Schekman, 1989; Lang et al., 2012; Ng et al., 1996). Sec63, on the other hand, influences both, post-translational and co-translational transport (Brodsky et al., 1995; Young, 2001). For the latter, Sec63 acts independently of Sec62 (Jermy et al., 2006; Mades et al., 2012). However, Sec63 is not essential in mammalian cells (Lang et al., 2012; Meyer et al., 2000; Tyedmers et al., 2000), since it could be functionally replaced by a similar protein Erj1 (Kroczynska et al., 2004). The mammalian homologue of Sec62 has gained a ribosome-binding site alluding to a possible contribution to co-translational transport (Müller et al., 2010). Sec62/Sec63 complex assembles in a 1:1 ratio with the core translocon (Meyer et al., 2000). Yeast has two additional subunits, Sec71 and Sec72 (Figure 5) in complex with Sec62/ Sec63 (Deshaies et al., 1991; Feldheim et al., 1993; Plath et al., 2004). Although mutations in sec71 or sec72 impair protein transport, they are not essential and their role is not understood (Fang & Green, 1994). BiP (binding immunoglobulin protein) BiP, also known as 78 kda glucose-regulated protein (GRP- 78), heat shock 70 kda protein 5 (HSPA5) or Kar2p in yeast, is an essential lumenal Hsp70-family chaperone. BiP has multiple functions during ER transport; it assists the insertion of pre-proteins into the Sec complex (Dierks et al., 1996), helps in gating the Sec complex (Alder et al., 2005; Hamman et al., 1998) and serves as a molecular ratchet during translocation (Nicchitta & Blobel, 1993; Tyedmers et al., 2003). Binding of BiP to the Sec complex occurs via its co-chaperone Sec63 (Lyman & Schekman, 1995, 1997) and Erj1 (Dudek et al., 2002). In addition, BiP has multiple lipid binding sites (Keller, 2011). Recently, the lumenal loop 7 of Sec61a was shown to contact BiP (Schäuble et al., 2012). Calmodulin The Sec61 pore is responsible for passive Ca 2+ efflux from the ER into the cytoplasm (Erdmann et al., 2011; Flourakis et al., 2006) and preventing Ca 2+ leakage requires channel gating. The chaperone BiP has been shown to seal the lumenal opening of the Sec61a during early translocation events (Alder et al., 2005; Hamman et al., 1998). In higher eukaryotes, Ca 2+ leakage is further reduced by the cytoplasmic protein calmodulin. Calmodulin is a universal mediator of Ca 2+ -controlled activity of numerous enzymes, ion channels, aquaporins and other proteins (Zhou et al., 2013). Recently, a high affinity binding site for calmodulin (IQ motif) was identified on the cytosolic N-terminus of Sec61a (Erdmann et al., 2011). Ca 2+ -bearing calmodulin binds to the translocon and limits ion flux (Erdmann et al., 2011) but the efficiency of calmodulin in restricting Ca 2+ leakage largely depends on the presence of BiP (Schäuble et al., 2012). Calmodulin might also have an additional role in the posttranslational targeting pathway (Shao & Hegde, 2011). TRAM (translocating chain associated membrane protein) TRAM is a 37-kDa glycoprotein that spans the ER membrane eight times with both N- and C-termini facing the cytoplasm (Tamborero et al., 2011). TRAM was identified as a major cross-linking partner of several secretory proteins in mammalian cells (Görlich et al., 1992; Krieg et al., 1989) and was shown to be required for protein transport in reconstituted proteoliposomes (Gorlich & Rapoport, 1993). Crosslinking data demonstrate that TRAM remains in contact with nascent chains even after their release from Sec61a (Liao et al., 1997; Sadlish et al., 2005). TRAM is suggested to act as a chaperone during the integration of less hydrophobic TM segments into the bilayer (Cross & High, 2009; Heinrich et al., 2000; Shao & Hegde, 2011). It is likely that TRAM cooperates with the Sec complex to assemble multiple TMs, a function similar to the proposed role of YidC during bacterial membrane insertion. TRAP (translocon associated protein complex) TRAP is a hetero-tetrameric protein complex that binds stoichiometrically to Sec61 (Hartmann et al., 1993; Ménétret et al., 2008). TRAP associates with Sec61 and oligosaccharyl transferase (OST, see below) to form the most abundant

12 68 K. Denks et al. Mol Membr Biol, 2014; 31(2 3): protein complexes associated with membrane-bound ribosomes (Potter & Nicchitta, 2002). In situ cryo electron tomography studies have mapped TRAP in complex with monomeric Sec61 (Pfeffer et al., 2012). TRAP accelerates transport of various substrates, but its precise function is unknown (Fons et al., 2003). Recent studies have suggested that TRAP is involved in the topogenesis of membrane proteins, affecting the translocation of charged residues (Sommer et al., 2013). OST (oligosaccharyl transferase) Approximately 70% of the eukaryotic secretome is potentially glycosylated (Zafar et al., 2011). Errors in glycosylation lead to misfolded proteins, giving rise to many congenital diseases (Schachter & Freeze, 2009). Asparagine-linked (N-linked) glycosylation takes place in the lumen of the ER and is catalyzed by OST, which transfers the dolichol-linked sugar unit to the corresponding sequon in the substrate protein (Tai & Imperiali, 2001). OST is a membrane-embedded heterooligomeric complex with at least eight different subunits in yeast (Karaoglu et al., 1997). The catalytic subunit STT 3 is highly conserved across the species (Burda & Aebi, 1999). N- glycosylation can occur co-translationally in a supramolecular complex of OST, the translating ribosome and Sec61 (Harada et al., 2009). OST binds to the ribosome near the tunnel exit (Harada et al., 2009) and to the Sec61 complex (Chavan et al., 2005; Pfeffer et al., 2014; Wang & Dobberstein, 1999). Shibatani et al. (2005) showed that mammalian OST complexes also have a high affinity to TRAM. N-linked glycosylation is one of the most common posttranslational modifications in eukaryotes, but is also conserved in several prokaryotes (Aebi et al., 2013; Baker et al., 2013). The catalytic subunit of the bacterial OST is called PglB and exhibits significant sequence similarity to the eukaryotic STT 3 (Schwarz & Aebi, 2011), however a direct interaction of PglB with the bacterial SecYEG complex remains to be demonstrated. Calnexin Calnexin is a type I ER membrane protein that serves as a constituent of the ER chaperone machinery for glycoproteins (Aebi et al., 2010). Calnexin can bind to its substrates both post-translationally and co-translationally, suggesting its proximity to the Sec61 translocon (Chen et al., 1995). A direct interaction between calnexin and the Sec61 complex was confirmed by two-hybrid analyses and co-immuneprecipitation (Boisramé et al., 2002). Recently, it was shown that palmitoylated calnexin is part of the ribosome-translocon complex and makes contact to the Sec61 associated TRAPa subunit (Lakkaraju et al., 2012a). Interestingly, the calnexinribosome-translocon complex appears to require the actin cytoskeleton for stabilization, which adds to the emerging concept that the cytoskeleton serves as an organizer and regulator of multiple cellular processes (Jaqaman & Grinstein, 2012; Kim & Coulombe, 2010). RAMP4 (ribosome-associated membrane protein) RAMP4 is a 7-kDa single-spanning membrane protein associating with the active ribosome-sec61 complex (Görlich et al., 1992). In cells with high secretory activity like hepatocytes, the unfolded protein response is induced in the absence of RAMP4 (Hori et al., 2006). Overexpression of RAMP4 in ER-stressed HEK293 cells supresses aggregation and degradation of newly synthesized membrane proteins (Yamaguchi et al., 1999). This indicates that RAMP4 is involved in membrane protein folding. RAMP4 has also been shown to regulate N-linked glycosylation of nascent secretory proteins (Lee et al., 2003; Schröder et al., 1999). Moreover, RAMP4 could be involved in the early sensing of a nascent chain since the eukaryotic ribosomal protein Rpl17 (E. coli L22 homologue) crosslinks to RAMP4 only if the nascent TM segment is buried inside the ribosomal tunnel (Pool, 2009). Erj1 ERj1 is a Sec63-related mammalian ER-membrane resident protein belonging to the Hsp40 family (Dudek et al., 2002). Erj1 contacts the ribosomal tunnel exit and BiP in the periplasm (Blau et al., 2005; Dudek et al., 2005), regulating protein translation in a BiP-dependent manner (Benedix et al., 2010; Dudek et al., 2005). Protein targeting to the Sec complex Signal sequences Signal peptides determine the cellular localization of proteins (Hegde & Bernstein, 2006). Proteins that are translocated via the Sec complex usually possess an N-terminal signal sequence, which has three parts: a positively charged N-terminal region, followed by a central hydrophobic H-region and a polar C-terminal region (Figure 7, (von Heijne, 1985). In secretory proteins, the C-region contains a cleavage site for signal peptidases (von Heijne, 1984). The general architecture of signal sequences is conserved, but they are highly variable in their primary sequence and length (von Heijne, 1986). Eukaryotic and prokaryotic signal sequences are interchangeable (von Heijne, 1985). Figure 7. The signal sequence. The signal peptides of the Sec translocon susbtrates in eukaryotes and prokaryotes share a common architecture, with a short, positively charged N-terminal region (N-region), a central, hydrophobic region (H-region) and a polar C-terminal region (C-region). The best conserved part of the signal peptide is the C-region, which can also contain a signal peptidase (SPase) cleavage site. This Figure is reproduced in color in the online version of Molecular Membrane Biology.