Involvement of PpiD in Sec-dependent protein translocation

The periplasmic space in between the inner and outer membrane of Gram-negative bacteria contains numerous chaperones that are involved in the biogenesis and rescue of extra-cytosolic proteins. In contrast to most of those periplasmic chaperones, PpiD is anchored by an N-terminal transmembrane domain within the inner membrane of Escherichia coli. There it is located in close proximity to the SecY subunit of the SecYEG translocon, which is the primary transporter for secretory and membrane proteins. By site-specific cross-linking we now found the periplasmic domain of PpiD also in close vicinity to the SecG subunit of the Sec translocon and we provide the first direct evidence for a functional cooperation between PpiD and the Sec translocon. Thus we demonstrate that PpiD stimulates in a concentration-dependent manner the translocation of two different secretory proteins into proteoliposomes that had been reconstituted with sub-saturating amounts of SecYEG. In addition we found ribosome-associated nascent chains of a secretory protein stalled at SecY also being in close contact to PpiD. Collectively these results suggest that PpiD plays a role in clearing the Sec translocon of newly translocated secretory proteins thereby improving the overall translocation efficiency. Consistent with this conclusion we demonstrate that PpiD contributes to the efficient detachment of newly secreted OmpA from the inner membrane and in doing so, seems to cooperate in a hierarchical manner with other periplasmic chaperones such as SurA, DegP, and Skp.     

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.     

YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.     

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.     

The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N‐terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin‐like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two‐state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease‐free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline‐containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.     

Involvement of SecDF and YidC in the membrane insertion of M13 procoat mutants

The M13 phage Procoat protein is one of the best characterized substrates for the novel YidC pathway. It inserts into the membrane independent of the SecYEG complex but requires the 60 kDa YidC protein. Mutant Procoat proteins with alterations in the periplasmic region had been found to require SecYEG and YidC. In this report, we show that the membrane insertion of these mutants also strongly depends on SecDF that bridges SecYEG to YidC. In a cold-sensitive mutant of YidC, the Sec-dependent function of YidC is strongly impaired. We find that specifically the SecDF-dependent mutants are inhibited in the cold-sensitive YidC strain. Finally, we find that subtle changes in the periplasmic loop such as the number and location of negatively charged residues and the length of the periplasmic loop can make the Procoat strictly Sec-dependent. In addition, we successfully converted Sec-independent Pf3 coat into a Sec-dependent protein by changing the location of a negatively charged residue in the periplasmic tail. Protease mapping of Pf3 coat shows that the insertion-arrested proteins that accumulate in the YidC- or in the SecDF-deficient strains are not translocated. Taken together, the data suggest that the Sec-dependent mutants insert at the interface of YidC and the translocon with SecDF assisting in the translocation step in vivo.     

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.     

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.     

Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase

The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.     

The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC.

KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions. The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop. The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD. To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region. The results show that also the first loop was translocated independently of YidC and the Sec translocase. We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases. When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC. To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments. Both constructed fragments, which contained KdpD-N (residues 1-448 of KdpD) and the KdpD-C (residues 444-894 of KdpD), readily inserted into the membrane. Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translocase and YidC. This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

YidC is involved in the biogenesis of the secreted autotransporter hemoglobin protease

Autotransporters (ATs) constitute an important family of virulence factors secreted by Gram-negative bacteria. Following their translocation across the inner membrane (IM), ATs temporarily reside in the periplasmic space after which they are secreted into the extracellular environment. Previous studies have shown that the AT hemoglobin protease (Hbp) of Escherichia coli requires a functional signal recognition particle pathway and Sec translocon for optimal targeting to and translocation across the IM. Here, we analyzed the mode of IM translocation of Hbp in more detail. Using site-directed photocross-linking, we found that the Hbp signal peptide is adjacent to YidC early during biogenesis. Notably, YidC is in part associated with the Sec translocon but has until now primarily been implicated in the biogenesis of IM proteins. In vivo, YidC appeared critical for the biogenesis of the ATs Hbp and EspC. For Hbp, depletion of YidC resulted in the formation of secretion-incompetent intermediates that were sensitive to degradation by the periplasmic protease DegP, indicating that YidC activity affects Hbp biogenesis at a late stage, after translocation across the IM. This is the first demonstration of a role for YidC in the biogenesis of an extracellular protein. We propose that YidC is required for maintenance of the translocation-competent state of certain ATs in the periplasm. The large periplasmic domain of YidC is not critical for this novel functionality as it can be deleted without affecting Hbp biogenesis.     

Identification of YidC Residues That Define Interactions with the Sec Apparatus

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.     

Investigating the SecY plug movement at the SecYEG translocation channel

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.     

SecYEG proteoliposomes catalyze the Deltaphi-dependent membrane insertion of FtsQ

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particle-dependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.     

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.     

Sec61beta--a component of the archaeal protein secretory system

Sec61p/SecYEG complexes mediate protein translocation across membranes and are present in both eukaryotes and bacteria. Whereas homologues of Sec61alpha/SecY and Sec61gamma/SecE exist in archaea, identification of the third component (Sec61beta or SecG) has remained elusive. Using PSI-BLAST, the archaeal counterpart of Sec61beta has been detected. With the identification of the Sec61beta motif, functions for a universal family of archaeal proteins can be predicted and the archaeal translocon system can be definitively detected.     

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway 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 hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.     

Polarity and Charge of the Periplasmic Loop Determine the YidC and Sec Translocase Requirement for the M13 Procoat Lep Protein

During membrane biogenesis, the M13 procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner with both the hydrophobic signal sequence and the membrane anchor sequence promoting translocation of the periplasmic loop via a hairpin mechanism. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. When the polarity of the translocated peptide region is lowered and the charged residues in this region are removed, translocation of this loop region occurs largely by a YidC- and Sec-independent mechanism. When the polarity is increased to that of the wild-type procoat protein, the YidC insertase is essential for translocation. Further increasing the polarity, by adding charged residues, switches the insertion pathway to a YidC/Sec mechanism. Conversely, we find that increasing the hydrophobicity of the transmembrane segments of PClep can decrease the translocase requirement for translocation of the peptide chain. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own.