Introduction
About one third of a typical proteome is exported to extracytoplasmic sites, emphasizing that protein translocation is a universal and crucial biological process. In nearly all organisms the vast majority of proteins is exported by the so-called Sec machinery. This represents an evolutionarily conserved protein complex embedded in the endoplasmic membrane of eukaryotes (Sec61) or the plasma membrane of prokaryotes (SecY) that functions as a protein-conducting channel, facilitating protein transport out of the cytoplasm as well as insertion of membrane proteins into the lipid bilayer (1). The Sec complex is made up of three different membrane proteins, namely a large subunit (Sec61/SecY) with ten transmembrane domains (TMDs) and two smaller subunits (Sec61β/ γ and SecE/G) that in most organisms span the membrane only once (1). The bacterial Sec system represents an excellent model for the analysis of protein translocation that has been studied for several decades with elegant genetic, biochemical and structural studies providing profound insight into the basic steps of Sec-dependent protein translocation. These are displayed in figure 1 (adopted from 2), showing that the translocation cycle begins with the sorting of substrates following or during their synthesis by ribosomes. Next, substrates are directed to the membrane by virtue of their N-terminal targeting sequence. This is recognized by different targeting factors and molecular chaperones that deliver the substrate at the membrane and keep it in a translocation-competent (unfolded) state. Subsequently, these proteins are translocated across the membrane by the Sec complex, while membrane proteins are inserted into the bilayer. The driving force for translocation is provided by different channel partners that associate with the Sec complex (1). Depending on the mode of translocation, these are either the ribosome or SecA. In cotranslational translocation, an elongating polypeptide chain moves directly from the translating ribosome into the SecY channel with the energy of the protein synthesis reaction probably as driving force for translocation. In posttranslational translocation, substrates are transported across the membrane following their synthesis. This is powered by SecA which is a conserved ATPase that is present in the cytosol of bacteria and the stroma of chloroplasts (1,2). This enzyme employs the energy of ATP hydrolysis to translocate polypeptides through the SecY channel in concert with the proton motive force (PMF). SecA is a multi-domain protein that can be viewed as a superfamily 2 RNA helicase specifically adapted for protein translocation with two nucleotide-binding domains (NBD1 and NBD2) that bind nucleotides at their interface. Moreover, SecA contains three other domains that are not found in other helicases, namely: the preprotein crosslinking domain (PPXD), helical wing domain (HWD), and helical scaffold domain (HSD) (3). Despite various bacterial SecA structures in different conformational states, it is not well understood how SecA drives protein translocation. However, several models have been put forward to explain its functioning with the power stroke model as most popular one. This proposes that the polypeptide is pushed through the Sec complex with ATP hydrolysis providing the energy for a power stroke and large domains of SecA would reach across the membrane to deliver the substrate at the other side (4). An improved version of this model was presented recently, which postulates that ATP binding and hydrolysis induce small conformational changes that result in a power stroke pushing the polypeptide forward in a stepwise fashion (5,6). Moreover, SecA is prominent in bacteria, including important human pathogens, while it is absent in humans. It, therefore, represents a potential target for the development of novel antimicrobials (7). A detailed molecular understanding of how SecA works is therefore crucial to design drugs that interfere with this pathway. With regards to this, it should be noted that recently detailed structures of the substrate-engaged SecA-SecY complex were presented (8). Here, I will discuss these structures as well as the current understanding of SecA functioning.
Overview of bacterial post-translational translocation
In all organisms proteins are translocated post-translationally, although its importance appears to be species-dependent. For example, in the Gram-negative bacterium E. coli about 27% of its proteome comprises proteins that are probably transported in a post-translational fashion outside of the cytoplasm (9), while in mammals the vast majority of proteins are translocated co-translationally (1). Despite a common translocation mode, post-translational export in bacteria is mechanistically different from its eukaryotic counterpart. Our knowledge about post-translational translocation in bacteria is based to a large extent on the work performed in E. coli. Biochemical and genetic experiments with this organism revealed that proteins destined for post-translational export are synthesized by cytosolic ribosomes initially as precursors with an N-terminal signal sequence (figure 2 adapted from 10) of about 18 to 30 residues. This signal sequence is organized into three distinct regions, namely: a positively charged N-terminal domain, a non-polar hydrophobic core and a more polar C-domain. The sequence of events during post-translational translocation (figure 2) was elucidated in detail by reconstituting the export reaction in vitro using purified E. coli translocation components and precursor proteins (11). This revealed that the nascent polypeptide chain associates with molecular chaperones such as trigger factor and DnaK that keep it in an unfolded state, while binding of SecB ensures cooperation with SecA and thereby targeting to the membrane-embedded SecY channel. The interaction of SecB and SecA require the signal sequence as well as the mature part of the precursor substrate. SecB, initially viewed as a targeting factor for post-translationally exported proteins, appears to play a broader role as molecular chaperone based on its interplay with trigger factor and DnaK. Therefore, SecB actively contributes to the cellular chaperone network that maintains protein homeostasis (12). Additionally, recent biochemical and structural data showed that SecA is also capable of binding directly to newly synthesized precursor proteins and interacts specifically with L23 on the large ribosomal subunit that serves as docking site for other chaperones and targeting factors. However, the mechanistic implication of these findings on the role of SecA in protein translocation is as yet not clear. Nevertheless, SecA delivers the substrate protein at the membrane embedded SecY channel, which triggers the release of SecB, thereby allowing SecB to rebind a newly synthesized substrate protein. Following the association of SecA with the translocon, SecA pushes the polypeptide through the channel using the energy of ATP hydrolysis to power the translocation of substrate proteins in a stepwise manner in which each catalytic cycle drives the translocation of a peptide segment of about 5 kDa. In addition to SecA, the PMF is also required to energize translocation. Although the role of the PMF is not well understood, it has been shown that SecA and the PMF act at different stages of the translocation cycle -ATP hydrolysis is required to initiate translocation, while the PMF can drive the late stages of protein translocation probably in the absence of SecA. Although SecY, SecE and SecG are sufficient for in vitro SecA powered protein translocation, the in vivo process involves SecD, SecF and YajC that bind to the SecYEG core. These proteins are not catalytically active and enhance in vitro protein translocation (13), while SecDF deficient E. coli cells are strongly impaired in protein export (14). These findings point towards a crucial role of SecD and SecF in translocation, although the precise mechanism is not clear. Conceivably, SecDF functions as membrane-embedded chaperone that is powered by the PMF to finalize the translocation reaction after the ATP dependent function of SecA (15). After completion of translocation, the signal sequence is proteolytically removed by a membrane-bound enzyme known as leader peptidase in the periplasm, while the mature protein folds into its biologically active conformation often with the help of periplasmic chaperones.
Biochemical and structural features of the bacterial post-translational translocation machinery
Structural features of SecA
Many of the components involved in bacterial protein export were discovered through genetic screens in E. coli aimed to identify: (i) suppressor mutants that restored protein translocation of protein substrates with defective signal sequences, or (ii) mutants that accumulated precursors of normally exported proteins in the cytosol (16). Using the latter approach, the gene encoding SecA (prlD)was identified in 1981, while its gene product was first characterized in 1982 (17,18). A role for SecA in protein translocation was unequivocally demonstrated in 1990 when the translocation reaction was reconstituted in vitro using purified SecA, SecY and SecE in liposomes (19). The same experimental setup was employed in 1997 to show that SecA comprises a unique translocation-specific ATPase that couples ATP hydrolysis to protein translocation (20). More recently, detailed atomic structures of different microbial components from the Sec machinery were solved, providing significant molecular insight into the mechanistic aspects of protein translocation (1). It is now well established that E. coli SecA is a cytosolic ATPase of about 102 kDa that binds peripherally to the plasma membrane, is essential for cell viability and displays affinity for SecYEG, SecB as well as precursor substrate proteins (21,2). Several lines of evidence indicate that SecA exists as a monomer or dimer, although the exact oligomeric organization during translocation has not been established yet and is highly debated (2). Most bacteria contain one SecA copy, while some Gram-positives and mycobacteria have been identified that contain two copies of the SecA protein: SecA1 and SecA2. It is now recognized that both proteins are functionally different with SecA1 responsible for the export of most proteins, whereas SecA2 only translocates a few proteins and probably plays a role in virulence (22).
Based on sequence similarity, SecA belongs to superfamily 2 of RNA helicases. Detailed crystal structures of SecA proteins from different bacteria are available. Figure 3 shows the structure of Thermotoga maritima SecA at 3.1Å (23) revealing that the protein is made up of two nucleotide-binding domains (NBD1 and NBD2) that are homologues to the ATPase site of superfamily 1 and 2 helicases. Both NBDs bind ATP at their interface, which gets hydrolyzed into ADP (in yellow spheres) resulting in conformational changes that drive protein translocation. Moreover, SecA is equipped with unique domains that enable it to interact with preproteins instead of RNA and facilitate their translocation. These include the preprotein cross-linking domain (PPXD) that represents the docking site for signal sequences. The PPXD is made up of α-helices and β-strands and is linked to NBD1. Additionally, the C-terminal part comprises two alpha helical subdomains, namely: (i) helical scaffold domain (HSD) and (ii) helical wing domain (HWD). The HSD (in magenta) is important for structural integrity (24) and contains a large helix that joins NBD2 and PPXD and therefore runs throughout the length of SecA. At its C-terminus, this helix is folded into a three-helix bundle. The HWD (in red) is inserted into this bundle and comprises two α-helices. The C-terminal end of SecA comprises a cysteine-rich zinc-binding domain that shows affinity for SecB and phospholipids (24). A surface representation of SecA is provided in the right panel of figure 3 and reveals a deep groove or clamp between PPXD and NBD2. It is thought that a precursor protein enters the open clamp and is subsequently captured by rotation of the PPXD, which closes the clamp (23). The structure that is shown in figure 3 therefore represents SecA in its open conformation.
Structural features of the substrate-engaged SecA-SecY complex
Despite the wealth of genetic and biochemical information on bacterial post-translational translocation, its full mechanistic understanding requires structures of the active SecY channel with translocation partners and a translocating polypeptide. It is therefore important to note that recently a cryo-EM structure of a translocating SecA SecY complex was presented at 3.5 Å (8). A surface (left panel) and ribbon representation (right panel) of this structure is provided in figure 4 and clearly reveals the pathway of a translocating polypeptide through SecA into the SecY channel and up to the lipid-embedded face of the Sec machinery. This complex was obtained by expressing in E. coli cells Bacillus subtilis SecA, Geobacillus thermodenitrificans SecYE as well as a substrate protein made up of the signal sequence of proOmpA linked to a GFP variant. In order to trap SecA in the state of ATP hydrolysis, the translocation complex was purified in the presence of ADP.BeFx, a nonhydrolyzable ATP analog mimicking ADP + Pi. Further, the translocating polypeptide was stabilized by a disulfide bridge, while the complex itself was stabilized by a nanobody against SecY. The structure of the active translocation complex shows SecY (in blue) and SecE (in yellow) with SecA (in green) at the cytosolic face. The translocating polypeptide chain is in blue. Importantly, the active SecY complex contains one SecA molecule, supporting the notion that this ATPase is physiologically active as a monomer. Within the complex, all subunits adopt previously reported structures (2). SecY contains 10 TMDs and can be divided into two halves comprising TMDs 1-5 and TMDs 6-10. These halves are stabilized by the TMD of SecE. When viewed from the side, the SecY channel represents an hourglass that is blocked by the presence of a short helix (part of TMD2), termed the plug (in pink, right panel). This domain probably moves towards the side during translocation, thereby opening the channel. At its center, the SecY channel contains a constriction containing six hydrophobic residues that form ring-like gasket (pore ring) around the translocating polypeptide, thereby preventing the leakage of ions and other small molecules. At the front, the translocation channel contains a gate between TMD 2 (in grey, right panel) and TMD 7 (in orange, right panel) that is used for the exit of hydrophobic targeting signals into the lipid bilayer. Within the right panel, NBD1 of SecA is in light pink, NBD2 in light blue, PPXD in green, HWD in light grey and HSD in dark pink, respectively.
SecA binds to SecY through different cytosolic loops (figure 5) and these include the loop between TMDs 6 and 7 (L6/7) and TMDs 8 and 9 (L8/9). The latter interacts with the PPXD and the C-terminal tail of SecA. Both SecY loops help to induce closure of the clamp between NBD2 and PPXD (figure 3), thereby ensuring that SecA adopts the closed conformation.
Figure 6 displays the pathway of the translocating polypeptide (in blue) from SecA into the SecY channel and all the way to exterior side of the translocation complex. The structure of SecY and E are omitted for clarity, while the domains of SecA are indicated. The signal sequence adopts a hairpin-like structure and is present near the exit of the lateral gate between TMDs 2 and 7 (right panel of figure 4) consistent with the results previously reported for other targeting sequences (2). The polypeptide following the signal sequence makes a U-turn (figure 6) and is located inside the translocation channel (figure 3) with the plug moved backwards and the residues of the pore ring forming a seal around it, thereby preventing the leakage of ions and other small molecules. Within the channel, the polypeptide adopts an extended conformation up to its entry point into SecA (figure 6). Here, the polypeptide is surrounded by the clamp that is formed by parts of the PPXD and HSD. The clamp associates with the substrate protein and induces a short β-strand in its polypeptide backbone. This interaction is sequence independently and may contribute to the ability of SecA to recognize a variety of polypeptides. A region of the polypeptide near the two-helix finger of SecA (THF) is not well resolved, suggesting that this part is flexible. The THF comprises a sub domain of the HSD made up of two helices that is inserted into the translocation channel (25). Several residues of the THF (in red spheres, right panel of figure 6) are close to the translocating polypeptide but don’t interact strongly with it. The cytosolic loops of SecY and the THF of SecA guide the polypeptide chain into the translocation channel, while the THF is responsible for pushing the polypeptide into the channel (6).
Mechanism of SecA powered translocation
Several models have been proposed to explain how SecA works. These include a ratcheting and a power stroke model. In the ratcheting model, the direction of the substrate protein through the SecY channel is determined by the ATPase activity of SecA that controls the opening of the channel, thereby preventing its backsliding and creating a net forward movement. In contrast, the power stroke model indicates that the polypeptide is pushed through the protein-conducting channel with ATP hydrolysis by SecA providing the energy for a power stroke. In the original model, large domains of SecA would reach across the membrane to deliver the substrate at the other side (4), while an improved version of this model was presented recently, which postulates that ATP binding and hydrolysis induce small conformational changes that result in a power stroke pushing the polypeptide forward in a stepwise fashion (5,6). Although the novel cryo-EM structure of the translocating SecA-SecY complex can’t distinguish between these two models, a recent study employing single molecule FRET experiments points towards a power stroke model (5). According to this study, the THF interacts with the polypeptide chain and pushes it into the translocation channel – the power stroke – upon ATP binding. In the cryo-EM structure, SecA is captured in the state of ATP hydrolysis with the THF interacting weakly with the substrate protein, while the clamp remains closed. The results of the aforementioned single molecule FRET study suggest that in this state the THF retracts from the polypeptide and the clamp is closed. The structural data are therefore consistent with the results of the single molecule FRET experiments. Conceivably, maintaining a closed clamp during retraction of the THF would prevent that the polypeptide is dragged backwards by the THF. It is thought that Pi release induces opening of the clamp via an outward rotation of SecY. Based on the results of the single molecule FRET study, a model for SecA powered translocation was proposed (figure 7 adopted from 5) that is supported by the novel cryo-EM structure of the translocating SecA-SecY complex. Translocation is initiated by ATP binding, inserting the THF into the SecY channel and thereby pushing the substrate protein towards the other side of the membrane (1). After this, the clamp closes around the polypeptide chain through a rotation of the PPXD and NBD2 (2). This could occur before or after ATP hydrolysis. During ATP hydrolysis (3), the THF retracts from the polypeptide, while the clamp remains closed. After ATP hydrolysis but before Pi is released, the THF has fully retracted (4), while the clamp is still closed. This allows the THF to return to its original position without dragging the polypeptide backwards. Following the release of Pi, the clamp reopens (5) and this allows the polypeptide to move freely in either direction. This passive sliding is an important part of the translocation mechanism as SecA is a major part of translocation cycle in the ADP bound state. In vivo, backsliding is probably restricted by the PMF, folding of the protein in the periplasm or association with periplasmic proteins.
Conclusion
Many proteins function outside of the cytoplasm, which requires their translocation across or insertion into the bacterial plasma membrane or eukaryotic endoplasmic membrane. In most cases, this is performed by an evolutionarily conserved protein-conducting channel termed the SecY complex in bacteria and Sec61 complex in eukaryotes (1). The export of proteins is powered by different translocation partners, which in bacteria are either the ribosome or SecA. This is a conserved ATPase that is present in the cytosol of bacteria and the stroma of chloroplasts. This enzyme couples ATP hydrolysis to post-translational protein translocation (2). In posttranslational translocation, substrates are transported across the membrane following their synthesis. Protein export through the bacterial Sec system has been studied for several decades with elegant genetic, biochemical and structural studies providing profound insight into the basic steps of Sec-dependent protein translocation (figure 2). However, a full mechanistic understanding of post-translational translocation requires structures of the active SecY channel with translocation partners and a translocating polypeptide. Moreover, SecA is prominent in bacteria, including important human pathogens, while it is absent in humans. It, therefore, represents a potential target for the development of novel antimicrobials (7). A detailed insight of how SecA works is therefore crucial to design drugs that interfere with post-translational translocation. Importantly, a cryo-EM structure of a translocating SecA SecY complex was presented recently that reveals the pathway of a translocating polypeptide through SecA into the SecY channel and up to the lipid-embedded face of the Sec machinery (8). This structure furthers the molecular understanding of how SecA powers the post-translational export of substrate proteins and cooperates with the SecY complex during this process. It can be expected that this increased understanding will contribute to the design of novel SecA inhibitors that impair bacterial protein export.
References
1. Rapoport TA, Li L, Park E. 2017. Structural and Mechanistic Insights into Protein Translocation. Annu Rev Cell Dev Biol. 33 :369-390.
2. Chatzi KE, Sardis MF, Karamanou S, Economou A. 2013. Breaking on through to the other side: protein export through the bacterial Sec system. Biochem J. 449: 25-37.
3. Hunt JF, Weinkauf S, Henry L, Fak JJ, McNicholas P, Oliver DB, Deisenhofer J. 2002. Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA. Science. 297: 2018-2026.
4. Economou A, Wickner W. 1994. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 78: 835-843.
5. Catipovic MA, Bauer BW, Loparo JJ, Rapoport TA. 2019. Protein translocation by the SecA ATPase occurs by a power-stroke mechanism. EMBO J. May 38(9). pii: e101140.
6. Erlandson KJ, Miller SB, Nam Y, Osborne AR, Zimmer J, Rapoport TA. 2008. A role for the two-helix finger of the SecA ATPase in protein translocation. Nature. 455: 984-987.
7. Chaudhary AS, Chen W, Jin J, Tai PC, Wang B. 2015. SecA: a potential antimicrobial target. Future Med Chem. 7: 989-1007.
8. Ma C, Wu X, Sun D, Park E, Catipovic MA, Rapoport TA, Gao N, Li L. 2019. Structure of the substrate-engaged SecA-SecY protein translocation machine. Nat Commun. 10(1):2872.
9. Song C, Kumar A, Saleh M. 2009. Bioinformatic comparison of bacterial secretomes. Genomics Proteomics Bioinformatics. 7 :37-46.
10. Rusch SL, Kendall DA. 2007. Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon. Biochim Biophys Acta. 1768: 5-12.
11. Hartl FU, Lecker S, Schiebel E, Hendrick JP, Wickner W. 1990. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell. 63 :269-279.
12. Sala A, Bordes P, Genevaux P. 2014. Multitasking SecB chaperones in bacteria. Front Microbiol. 5:666.
13. Duong F, Wickner W. 1997. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J. 16: 2756-2768.
14. Pogliano JA, Beckwith J. 1994. SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 13: 554-561.
15. Tsukazaki T1, Mori H, Echizen Y, et al. 2011. Structure and function of a membrane component SecDF that enhances protein export. Nature. 474: 235-238.
16. Benson SA, Hall MN, Silhavy TJ. 1985. Genetic analysis of protein export in Escherichia coli K12. Annu Rev Biochem. 54: 101-34.
17. Oliver DB, Beckwith J. 1981. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 25: 765-772.
18. Oliver DB, Beckwith J. 1982. Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli. J Bacteriol. 150: 686-691.
19. Brundage L, Hendrick JP, Schiebel E, Driessen AJ, Wickner W. 1990. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 62: 649-57.
20. Lill R, Dowhan W, Wickner W. 1990. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 60: 271-280.
21. Cabelli RJ, Chen L, Tai PC, Oliver DB. 1988. SecA protein is required for secretory protein translocation into E. coli membrane vesicles. Cell. 55: 683-692.
22. Feltcher ME, Braunstein M. 2012. Emerging themes in SecA2-mediated protein export. Nat Rev Microbiol. 10 :779-789.
23. Zimmer J, Rapoport TA. 2009. Conformational flexibility and peptide interaction of the translocation ATPase SecA. J Mol Biol. 394: 606-612.
24. Hunt JF, Weinkauf S, Henry L, Fak JJ. et al. 2002. Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA. Science. 297: 2018-2026.
25. Zimmer J, Nam Y, Rapoport TA. 2008. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature. 455: 936-943.
Reactie schrijven