3.A.5 The General Secretory Pathway (Sec) Family
Protein secretion in bacteria can be achieved by an ABC-type transport system (the Type I protein secretion system, TC #3.A.1), the general secretory pathway (the Sec or Type II protein secretion system described here), and three additional types of systems (the Type III, Type IV and Tat protein secretion systems, TC #3.A.6, 3.A.7 and 2.A.64, respectively). Protein complexes of the Sec family are found universally in prokaryotes and eukaryotes. The translocase in E. coli consists of three integral inner membrane proteins, SecYEG, and the cytoplasmic ATPase, SecA. SecA recruits SecYEG complexes to form the active translocation channel (Collinson et al., 2001). A long α-helix in SecA is important for coupling of ATPase activity to protein translocation (Mori and Ito, 2006). The rate limiting step, ADP dissociation from SecA, is accompanied by a structural rearrangement that is strongly coupled to the protein interface and protein translocation through SecYEG (Robson et al., 2009). Signal peptides are allosteric activators of the protein translocase (Gouridis et al., 2009). The assembly of bacterial inner membrane proteins by the Sec and YidC proteins has been reviewed (Dalbey et al., 2011; Kudva et al. 2013).
A structure of a SecA-SecY complex raises the possibility that the polypeptide chain is moved by a two-helix finger domain of SecA that is inserted into the cytoplasmic opening of the SecY channel. Erlandson et al., 2008 have used disulphide-bridge crosslinking to show that the loop at the tip of the two-helix finger of E. coli SecA interacts with a polypeptide chain right at the entrance into the SecY pore. Mutagenesis demonstrated that a tyrosine in the loop is particularly important for translocation, but it can be replaced by some other bulky, hydrophobic residues. They proposed that the two-helix finger of SecA moves a polypeptide chain into the SecY channel with the tyrosine providing the major contact with the substrate, a mechanism analogous to that suggested for hexameric protein-translocating ATPases. A non-proteinaceous 'glycolipozyme' called membrane protein integrase, MPIase, has been reprorted to facilitate Sec-dependent protein translocation and membrane protein insertion as well as SecG inversion (Moser et al. 2013).
The E. coli SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. Helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10 (Lycklama et al., 2010). Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants, translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggests that only a limited movement of the plug domain suffices for translocation.
Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids. While the cytoplasmic cavity is filled with the plug, which moves out of the way during protein translocation. The channel transports large polypeptides and yet prevents the passage of small molecules, such as ions and metabolites. In the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules (Park and Rapoport, 2011). The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained during protein translocation.
Osborne and Rapoport (Osborne and Rapoport 2007) showed that the two SecYEG monomers in the dimeric complex each plays a distinct role. One SecY complex serves as the protein-conducting channel, whereas its non-translocating counterpart forms a static docking site for the ATPase motor domain of SecA. This model is consistent with results showing that a single SecY complex is sufficient to bind to SecA. Taking into consideration the dimensions of the SecY dimer and SecA as well as the deep groove observed in the crystal structure of SecA, the authors postulated that this groove is involved in binding to signal sequences and polypeptide chains. And, it is located just below the active copy of SecY in the channel. This would be an optimal position for pushing the protein substrate through. (Duong, 2007). SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides 'friction' that minimizes backsliding when ADP-bound SecA resets to 'grab' the next segment of the substrate (Erlandson et al., 2008). Deville et al. (2011) have addressed the functional significance of the oligomeric status of SecYEG in protein translocation. While monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. However, Kedrov et al. (2011) have reported that a single copy of SecYEG is sufficient for preprotein translocation.
The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane (Ménétret et al., 2007). Nontranslating E. coli ribosome binds to a single SecY complex. Two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. Point mutations of basic residues within either loop abolish ribosome binding. Ménétret et al. (2007) suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain. It has been reported that Sec translocons can accomodate at least two hydrophilic translocating peptides that are separated by multiple hydrophobic TMSs (Skach, 2007; Kida et al., 2007). Depending on channel binding partners, polypeptides are moved through the heterotrimeric Sec channel by 3 different mechanisms: the polypeptide chain is transferred dircetly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechamism is used by the bacterial ATPase SecA (Rapoport, 2007). Sec-dependent protein translocation across the endoplasmic reticulum membranes of eukaryotes has been reviewed (Zimmermann et al., 2010).
In eukaryotes, the corresponding Sec61p complex forms a dynamic precursor-activated channel (Wirth et al., 2003). The channel activity has been reconstituted in artificial lipid bilayers and exhibits conductances corresponding to channel openings of from 6 to 60 Å (Wirth et al., 2003). Several proteins, named according to the complex (Sec or SRP) and the protein size (in kDa), comprise the yeast Sec-SRP complex (see TC entry #3.A.5.8.1) and the mammalian Sec-SRP complex (see TC entry #3.A.5.9.1) (Wild et al., 2004). Yet1p and Yet3p, the yeast homologs of BAP29 and BAP31, interact with the endoplasmic reticulum translocation apparatus (Wilson & Barlowe et al., 2010). Wilson & Barlowe (2010) have suggested that Yet1 and Yet3 reside at the translocation pore to manage biogenesis of specific transmembrane secretory proteins. In eukaryotes, the ER lumenal molecular chaperone, BiP, contributes to efficiency and unidirectional transport (Zimmermann et al., 2011).
Janda et al., (2010) produced a fusion protein between Sulfolobus solfataricus SRP54 (Ffh) and a signal peptide connected via a flexible linker. This fusion protein oligomerized in solution through interaction between the SRP54 and signal peptide moieties. It was functional, as demonstrated by its ability to bind SRP RNA and the SRP receptor, FtsY. A crystal structure at 3.5 A resolution of an SRP54-signal peptide complex in the dimer, reveals how a signal sequence is recognized by SRP54. Seppala et al., (2010) reported that the topology of an E. coli inner membrane protein with four or five transmembrane helices could be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C terminus. This observation points to an unanticipated plasticity in membrane protein insertion mechanisms (Seppälä et al., 2010).
Most bacteria have only one set of Sec proteins (e.g. SecY, SecE, SecG and SecA). However, in some Gram-positive bacteria (Streptococcus gordonii, S. pneumoniae and Staphylococcus aureus) there are two copies of these proteins. The second copy (SecY2, SecA2 and Asp1-5 in S. gordonii) comprise a specialized system for the transport of very large, serine-rich, cell surface, repeat proteins (e.g., the serine-rich platelet-binding protein, GspB [AAL13053; 3072 aas] in S. gordonii), that are important for pathogenesis (Bensing and Sullam, 2002; Bensing et al., 2004; Takamatsu et al., 2004, 2005). Asp4 and 5 may be the functional homologues of SecE and SecG, respectively (Takamatsu et al., 2005). This is the only known example of sec gene duplication in the prokaryotic world. Glycine residues in the hydrophobic core of the substrate adhesin, GspB, signal sequence route export towards the accessory secretory pathway and away from the general secretory pathway (Bensing et al., 2007). Some bacteria, such as the Mycobacterial species, have two SecA proteins (SecA1 and SecA2) where SecA2 serves as a part of a specialized protein export system such as for sugar binding lipoproteins (Gibbons et al., 2007).
Two auxiliary proteins, SecD and SecF in E. coli (a single protein in Bacillus subtilis), are together homologous to members of the RND superfamily (TC #2.A.6). Another protein, YajC of E. coli, forms a complex with SecD-SecF both independently of and in complexation with SecYEG. The SecDF-YajC complex is not essential for secretion but stimulates secretion up to ten-fold under many conditions, particularly at lower temperatures. Tsukazaki et al. (2011) determined the crystal structure of the Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and the proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. They proposed that SecDF functions as a membrane-integrated chaperone, powered by the proton motive force, to achieve ATP-independent protein translocation.
SecY is a 10 TMS protein of about 450 amino acyl residues that forms the protein translocating channel. Two smaller integral membrane proteins, SecE and SecG, each about 140 amino acyl residues in length, are found complexed with SecY. Translocation is driven by ATP hydrolysis catalyzed by the SecA ATPase constituent of the translocase which associates tightly with SecY. Both SecY and SecA directly contact the substrate protein. Although protein export is driven by ATP hydrolysis, the pmf is stimulatory. Possibly both energy sources are required for efficient translocation, with each acting at different steps (van der Laan et al., 2004). Point mutations in SecY abolish the pmf-dependence of the translocation process, but ATP hydrolysis is essential under all conditions. Sugai et al., (2007) have suggested that SecG undergoes topological inversion, and that this inversion is essential for SecA-dependent stimulation of protein translocation.
The archaeal (M. jannaschii) SecYEG complex in an inactive state has been solved to 3.2 Å resolution. The structure suggests that one copy of the heterotrimer may serve as the functional translocation channel (van den Berg et al., 2004). SecY (β-subunit) has two linked halves (TMSs 1-5 and 6-10), forming a 'clam shell.' One side is hinged by the loop between TMSs 5 and 6; they are clamped together by SecE (the β-subunit). The other side is unconstrained and forms the lateral gate. A cytoplasmic funnel leading into the channel is plugged by a short helix (TMH2a). Plug displacement opens the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction that may form a temporary seal. The structure leads to a suggestion as to how TMSs exit laterally into the lipid bilayer (van den Berg et al., 2004). Feautures of TMSs that promote lateral release from the translocase into the lipid bilyaer have been defined (Xie et al., 2007).
The SecY proteins of archaea and the Sec61 proteins in the endoplasmic reticula of Saccharomyces cerevisiae and other eukaryotes show sequence similarity to and are homologous to SecY of E. coli and other bacteria. The SecA proteins of bacteria show some sequence similarity to a region of the SRP72 protein of Homo sapiens. The Sec system can both translocate proteins across the cytoplasmic membrane and insert integral membrane proteins into it. The former proteins but not the latter proteins possess N-terminal, cleavable, targeting signal sequences that are required to direct the proteins to the Sec complex.
The transmembrane α-helices in integral membrane proteins can be recognized co-translationally and inserted into the ER membrane by the Sec61 translocon. Using in vitro translation of a model protein in the presence of dog pancreas rough microsomes, Hessa et al. (2007) analyzed a large number of systematically designed hydrophobic segments. They analyzed the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, and also determined the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer (Hessa et al., 2007).
Recent cryo-electron microscopy of the active E. coli SecYEG complex with a translating ribosome has revealed structural aspects of the functional, cotranslational:translocational complex (Mitra et al., 2005). The results favor a front-to-front arrangement of two SecYEG complexes in the protein-conducting channel. They support channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of the observation in the translocating protein-conducting channel of two segregated pores with different degrees of access to bulk lipid, a model for co-translational protein translocation was proposed (Mitra et al., 2005).
The SecY-Sec61 phylogenetic tree reveals ten clusters according to organismal phylogeny as follows: (1) four clusters from Gram-negative bacteria (proteobacteria, spirochetes, chlamydia and primitive bacteria), (2) two clusers from Gram-positive bacteria (high and low G+C organisms), (3) Mycoplasma, (4) cyanobacteria and eukaryotic chloroplasts, (5) archaea, (6) eukaryotes. Only the SecY-Sec61 constituents are indicated in the table of proteins below, except for the well-characterized E. coli, Saccharomyces cerevisiae and Homo sapiens systems (Cao and Saier, 2003).
In eukaryotes, the heterotrimeric Sec61 protein complex in the endoplasmic reticulum (ER) serves as the channel for protein transport by either a cotranstranslational or posttranslational mechanism. In cotranslational export, directionality is determined by binding of the translating ribosome to the Sec61 complex. The channels in the ribosome and membrane are aligned so the luminal end of the channel is the only exit site available to the elongating polypeptide chain. By contrast, in posttranslational transport, the Sec61 complex associates with the tetrameric Sec62/63 complex, the resultant Sec complex binds the signal sequence of the translocation substrate, and translocation is energized by BiP (Kar2), a soluble, luminal Hsp70 ATPase that hydrolyzes ATP to translocate polypeptides. Translocation requires that BiP interacts with the Sec complex via a luminal domain of Sec63, the J domain. BiP may 'pull' the protein through the channel and/or act as a 'molecular ratchet', preventing backward movement. While both mechanisms may be operative, the ratchet mechanism is clearly operative under certain conditions (Matlack et al., 1999; Misselwitz et al., 1998).
Some evidence suggests that the ER translocon can function as a 'retrotranslocon' to transport improperly folded proteins from the lumen of the ER, back into the cytoplasm where degradation occurs in proteosomes. Thus, ER lumen proteins that are stalled at some point in their folding/assembly, and possibly integral membrane proteins that do not properly fold, may be recognized by specific chaparone proteins and targeted for retrotranslocation (Johnson and Haigh, 2000). The process requires cytoplasmic proteins and ATP. In one case, that of the cholera toxin Al chain, protein disulfide isomerase acts as a redox-dependent unfoldase, feeding the toxin into the Sec61 complex for retrotranslocation (Tsai et al., 2001). More recent work suggests that a distinct complex of proteins may be involved (see the ER-RT family; TC #3.A.16, and Lilley and Ploegh, 2004; Ye et al., 2004).
The SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes (1) a Prl-like phenotype, (2) reduced proton-motive force dependence of translocation, (3) increased membrane insertion of SecA, (4) diminished requirement for functional leader peptide, and (5) weakened SecYEG subunit association. Locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. It may contribute to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state (Maillard et al., 2007).
Insertion of integral inner membrane proteins in bacteria is dependent on a complex resembling the eukaryotic SRP protein-RNA complex. These proteins (Ffh; an SRP-like protein) and FtsY (an SRP receptor β-subunit-like protein) probably act like chaparones, feeding into the Sec system. Insertion of most polytopic inner membrane proteins shows a dependency on Ffh, the 4.5S RNA molecule and FtsY. Compaction of prokaryotic signal-anchor transmembrane domains begins within the ribosome tunnel but is stabilized by SRP during targetting (Robinson et al. 2012). The GTPases of SRP and its receptor are mutually stimulated by their interaction (Egea et al., 2004). They form a quasi-two fold symmetrical heterodimer. SRP-dependent protein insertion appears to be dependent on the SecYEG channel complex. An involvement of FtsE has been proposed where FtsE and FtsY work together as an ABC-type protein insertion system, inserting K+ pump proteins into the membrane of E. coli (Ukai et al., 1998). This possibility should not be considered as proven. FtsY contains a conserved autonomous lipid-binding amphipathic alpha-helix at its N-terminal end. This helix is essential for FtsY function in vivo, and mediates the physiologically relevant interaction of FtsY with lipids (Parlitz et al., 2007; Braig et al., 2011). Signal sequence-independent SRP-SR complex formation at the membrane suggests alternative targeting pathways within the SRP cycle (Braig et al., 2011).
During the biogenesis of hydrophobic inner membrane proteins in E. coli, at least three mechanistically different modes of integration can be distinguished as follows: (i) signal recognition particle (SRP) (comprising Ffh and 4.5 S RNA) and SR (SRP receptor FtsY)-dependent targeting to the SecYEG translocon; (ii) SRP/SR- and SecYEG-dependent integration assisted by the translocation ATPase SecA to translocate hydrophilic domains of composite membrane proteins; and (iii) SRP- and Sec-independent integration of several small membrane proteins or subdomains thereof. Some soluble secreted protein lacking signal sequences are translocated into the periplasm by a SRP-dependent pathway (Ren et al., 2007).
In contrast to the original idea of a spontaneous process, the integration of those small SRP- and Sec-independent membrane proteins depends on the inner membrane YidC protein (2.A.9.3.1). It has been proposed that in these cases YidC fulfils the function of an 'insertase' on its own. In addition to this essential function, YidC seems to play a role as membrane chaperone for more complex membrane proteins by assisting in the release of transmembrane helices from the SecYEG translocon, in helix packing, and in the proper folding of polytopic membrane proteins.
E. coli synthesize over 60 small proteins of less than 50 amino acids with 65% containing a single predicted TMS domain (Fontaine et al., 2011). Examples of both N(in)-C(out) and N(out)-C(in) orientations were found in assays of topology-reporter fusions, but three of nine tested proteins display dual topology. The positive inside rule applies. Membrane insertion is generally dependent on the Sec and YidC proteins.
The mechanism of integration of the polytopic E. coli membrane protein mannitol permease (MtlA) involves targeting to the inner membrane by SRP/SR, followed by its co-translational integration into the lipid bilayer via the SecYEG translocon. SecA and SecG are dispensable in accordance with the fact that MtlA possesses no large periplasmic regions. Nascent MtlA chains efficiently cross-link to YidC. The minimal integration machinery consists of SecYEG and a novel integration-stimulating factor in the inner membrane of E. coli. This factor, which also stimulated the integration of the M13 procoat protein in the absence of any other protein if spontaneous integration was prevented is a lipid A-derived compound (Nishiyama et al., 2006).
E. coli SRP targets membrane proteins into the inner membrane after binding translating ribosomes. When a signal sequence emerges, SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. A 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor reveals the regions that are involved in complex formation, provide insight into the conformation of SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence (Schaffitzel et al., 2006).
The signal sequences of two surface proteins in Streptococcus pyogenes, M protein and protein F (PrtF), direct secretion to different subcellular regions (Carlsson et al., 2006). The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old septum. Thus, a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. An increased level of complexity in protein translocation is suggested (Carlsson et al., 2006).
The Gram-positive pathogen Streptococcus pyogenes secretes proteins through the ExPortal, a unique single microdomain of the cellular membrane specialized to contain the Sec translocons (Rosch and Caparon, 2005). It has been proposed that the ExPortal functions as an organelle to promote the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and membrane-associated chaperones. HtrA (DegP), a surface anchored accessory factor, is required for maturation of the secreted SpeB cysteine protease, localized exclusively to the ExPortal. Furthermore, the ATP synthase β subunit is not localized to the ExPortal, suggesting that retention is likely restricted to a specific subset of exported proteins. Mutations that disrupted the anchoring, but not the protease activity of HtrA, also altered the maturation kinetics of SpeB, demonstrating that localization to the ExPortal is important for HtrA function. The ExPortal provides a mechanism by which Gram-positive bacteria coordinate protein secretion and subsequent biogenesis in the absence of a specialized protein-folding compartment.
A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms; (1) the polypeptide chain is transferred directly into the channel by the translating ribosome, (2) a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and (3) a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data suggest how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lapid bilayer, and maintains the membrane barrier for small molecules (Rapoport, 2007). The lateral translocon gate in Sec61 is formed by four TMSs (TM2, TM3, TM7, and TM8) (Trueman et al., 2011).
The Sec secretory pathway functions in transport of proteins across the cytoplasmic membrane. Then a membrane-anchored periplasmic chaperone, PpiD, accepts the translated substrate protein from the SecYEG channel and folds it (Antonoaea et al., 2008). A distinct protein complex, termed the main terminal branch (MTB; TC #3.A.15), is responsible for exoprotein secretion across the outer membrane of a wide variety of Gram-negative bacteria.
The general secretory system of E. coli translocates polypeptides that are devoid of stable tertiary structure; precursors must be bound by components of the export apparatus before they fold. In some cases, the nonnative precursor can be captured directly by SecA, but often, SecB, a cytosolic chaperone (molar mass, 69 kDa), first interacts with the ligand. Subsequently, a ternary complex is formed by binding SecA. SecA has high affinity for SecY, and the precursor is delivered to the translocon at the membrane. When SecA, carrying a precursor binds the translocon, its ATPase activity is stimulated to couple the energy of hydrolysis to mechanical work which moves the precursor through the channel. In vivo, once the process has been started, translocation can be completed by the energy of protonmotive force (Cooper et al., 2008).
Movement of the precursor through the channel occurs in multiple, successive steps while SecA undergoes cycles of binding and hydrolysis of ATP. The transduction of the chemical energy to mechanical work involves conformational changes in SecA. Copper et al. (Copper et al. 2008) provided a detailed description of the interactions between the binding partners and the molecular movements that result from such interactions. Signal peptides are allosteric activators of the protein translocase (Gouridis et al., 2009).
The SecA ATPase forms a functional complex with the protein-conducting SecY channel to translocate polypeptides across the bacterial cell membrane. SecA recognizes the translocation substrate and catalyzes its unidirectional movement through the SecY channel. The crystal structure of the Thermotoga maritima SecA-SecYEG complex showed the ATPase in a conformation where the nucleotide-binding domains (NBDs) have closed around a bound ADP-BeFx complex, and SecA's polypeptide-binding clamp is shut. Zimmer and Rapoport (2009) presented the crystal structure of T. maritima SecA in its ADP-bound form at 3.1 A resolution. SecA alone has a different conformation in which the nucleotide-binding pocket between NBD1 and NBD2 is open and the preprotein cross-linking domain has rotated away from both NBDs, thereby opening the polypeptide-binding clamp. They also determined a structure of the Bacillus subtilis SecA in complex with a peptide at 2.5 A resolution. This structure showed that the peptide augments the highly conserved beta-sheet at the back of the clamp. These structures suggest a mechanism by which ATP hydrolysis can lead to polypeptide translocation (Zimmer and Rapoport, 2009).
The transmembrane domains in a membrane protein must be recognized and correctly oriented before their insertion into the lipid bilayer (Shao and Hegde, 2011). Devaraneni et al. (2011) generated 'snapshots' at different stages of membrane protein biogenesis, revealing a dynamic set of steps that imply an unexpectedly flexible membrane insertion machinery. Four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the ribosome-Sec61 translocon complex (RTC), (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. A canonical type II signal anchor (SA) inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment (Devaraneni et al., 2011).
Proteolytic cleavage of proteins that are permanently or transiently associated with the cytoplasmic membrane is crucially important for a wide range of essential processes in bacteria. This applies to the secretion of proteins and to membrane protein quality control. The responsible membrane proteases include signal peptidases, signal peptide hydrolases, FtsH, the rhomboid protease GlpG, and the site 1 protease DegS in E.coli. These enzymes employ very different mechanisms to cleave substrates at the cytoplasmic and extracytoplasmic membrane surfaces or within the plane of the membrane. Dalbey et al. 2012 describe the different ways that bacterial membrane proteases degrade their substrates, emphasizing the catalytic mechanisms of substrate delivery to the respective active sites.
The IISP complex catalyzes two-step protein export as follows:
ATP + pmf + unfolded protein (cytoplasm) ⇌ ADP + Pi + unfolded protein
(periplasm or cytoplasmic membrane)