Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.     

The Sec translocon mediated protein transport in prokaryotes and eukaryotes

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/Sec61α 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.     

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.     

Cytoplasmic targeting of IpaC to the bacterial pole directs polar type III secretion in Shigella

Type III secretion (T3S) systems are largely used by pathogenic gram-negative bacteria to inject multiple effectors into eukaryotic cells. Upon cell contact, these bacterial microinjection devices insert two T3S substrates into host cell membranes, forming a so-called 'translocon' that is required for targeting of type III effectors in the cell cytosol. Here, we show that secretion of the translocon component IpaC of invasive Shigella occurs at the level of one bacterial pole during cell invasion. Using IpaC fusions with green fluorescent protein variants (IpaCi), we show that the IpaC cytoplasmic pool localizes at an old or new bacterial pole, where secretion occurs upon T3S activation. Deletions in ipaC identified domains implicated in polar localization. Only polar IpaCi derivatives inhibited T3S, while IpaCi fusions with diffuse cytoplasmic localization had no detectable effect on T3S. Moreover, the deletions that abolished polar localization led to secretion defects when introduced in ipaC. These results indicate that cytoplasmic polar localization directs secretion of IpaC at the pole of Shigella, and may represent a mandatory step for T3S.     

Penetration into membrane of amino‐terminal region of SecA when associated with SecYEG in active complexes

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino‐terminal region of SecA with membrane. We use site‐directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co‐assembled into lipids with SecYEG to yield highly active translocons, the N‐terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N‐terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N‐terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.     

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein‐conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross‐) correlation spectroscopy with single‐molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.     

Membrane protein insertion of variant MscL proteins occurs at YidC and SecYEG of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously.     

Topology of the SecA ATPase Bound to Large Unilamellar Vesicles

The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.     

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.     

SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri

The spreading ability of Shigella flexneri , a facultative intracellular Gram-negative bacterium, within the host-cell cytoplasm is the result of directional assembly and accumulation of actin filaments at one pole of the bacterium. IcsA/VirG, the 120 kDa outer membrane protein that is required for intracellular motility, is located at the pole of the bacterium where actin polymerization occurs. Bacteria growing in laboratory media and within infected cells release a certain proportion of the surface-exposed IcsA after proteolytic cleavage. In this study, we report the characterization of the sopA gene which is located on the virulence plasmid and encodes the protein responsible for the cleavage of IcsA. The deduced amino acid sequence of SopA exhibits 60% identity with those of the OmpT and OmpP outer membrane proteases of Escherichia coli . The construction and phenotypic characterization of a sopA mutant demonstrated that SopA is required for exclusive polar localization of IcsA on the bacterial surface and proper expression of the motility phenotype in infected cells.     

Dropping Out and Other Fates of Transmembrane Segments Inserted by the SecA ATPase

Type II single-span membrane proteins, such as CadC or RodZ, lacking a signal sequence and having a far-downstream hydrophobic segment, require the SecA secretion motor for insertion into the inner membrane of Escherichia coli. Using two chimeric single-span proteins containing a designed hydrophobic segment H, we have determined the requirements for SecA-mediated secretion, the molecular distinction between TM domains and signal peptides, and the propensity for hydrophobic H-segments to remain embedded within the bilayer after targeting. By means of engineered H-segments and a strategically placed SPase I cleavage site, we determined how targeting and stability of the chimeric proteins are affected by the length and hydrophobicity of the H-segment. Very hydrophobic segments (e.g., 16 Leu) are stably incorporated into the inner membrane, resulting in a C-terminal anchored membrane protein, while a 24L construct was not targeted to the membrane by SecA and remained in the cytoplasm. However, a construct carrying preMalE at the N-terminus led to SecA targeting to SecYEG via the native signal sequence and stable insertion of the downstream 24L H-segment. We show that the RseP intramembrane protease degrades weakly stable H-segments and is a useful tool for investigating the borderline between stable and unstable TM segments. Using RseP⁻ cells, we find that moderately hydrophobic sequences (e.g., 5Leu + 11Ala) are targeted to SecYEG by SecA and inserted, but subsequently drop out of the membrane into the cytoplasm. Therefore, the free energy of transfer from translocon to bilayer is different from the transfer free energy from membrane to water.     

Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA

The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal beta domain forms a beta barrel channel in the outer membrane through which the amino-terminal alpha domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.     

The oligomeric state and arrangement of the active bacterial translocon

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that 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. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.     

SecA-mediated targeting and translocation of secretory proteins

More than 30 years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder–order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Identification of two interaction sites in SecY that are important for the functional interaction with SecA

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.     

Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB

In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.