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Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.

ABSTRACT: During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

SUBMITTER: Wu ZC

PROVIDER: S-EPMC3318685 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.

Wu Zht Cheng ZC de Keyzer Jeanine J Kedrov Alexej A Driessen Arnold J M AJ

The Journal of biological chemistry 20120120 11

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane ...[more]

PMID: 22267723

Similar Datasets

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon.

Project description:Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.

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The Dynamic SecYEG Translocon.

Project description:The spatial and temporal coordination of protein transport is an essential cornerstone of the bacterial adaptation to different environmental conditions. By adjusting the protein composition of extra-cytosolic compartments, like the inner and outer membranes or the periplasmic space, protein transport mechanisms help shaping protein homeostasis in response to various metabolic cues. The universally conserved SecYEG translocon acts at the center of bacterial protein transport and mediates the translocation of newly synthesized proteins into and across the cytoplasmic membrane. The ability of the SecYEG translocon to transport an enormous variety of different substrates is in part determined by its ability to interact with multiple targeting factors, chaperones and accessory proteins. These interactions are crucial for the assisted passage of newly synthesized proteins from the cytosol into the different bacterial compartments. In this review, we summarize the current knowledge about SecYEG-mediated protein transport, primarily in the model organism Escherichia coli, and describe the dynamic interaction of the SecYEG translocon with its multiple partner proteins. We furthermore highlight how protein transport is regulated and explore recent developments in using the SecYEG translocon as an antimicrobial target.

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The SecYEG interactome of E. coli: non-competitive binding of PpiD and YidC to the SecYEG translocon

Project description:The SecYEG translocon constitutes the major protein transporting channel in bacteria and transports an enormous variety of different secretory and inner membrane proteins. The minimal core of the SecYEG translocon consists of the three inner membrane proteins SecY, SecE and SecG, which together with appropriate targeting factors are sufficient for protein transport in vitro. However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, which likely allow the SecYEG translocon to manage diverse substrates. For obtaining a global view on SecYEG plasticity, a label free proteomics approach was executed, which identified known SecYEG interacting proteins, verified the interaction with quality control proteins and identified several novel putative SecYEG interactors. Surprisingly, the chaperone complex PpiD/YfgM was identified as the most pronounced contact partner of SecYEG and was much dominant than the established partner protein YidC. Detailed analyses of the PpiD and YidC contact sites on SecY by site directed cross-linking revealed that PpiD and YidC use almost completely overlapping binding sites on SecY and contact the lateral gate, the plug domain and the periplasmic cavity of SecY. Still, despite having almost identical binding sites, their binding to SecY is non-competitive as determined by quantitative mass spectrometry and cross-linking. This implies that the SecYEG translocon forms different substrate-independent sub-assemblies, in which SecYEG is either in contact with YidC or with the PpiD/YfgM complex.

2019-11-07 | PXD015974 | Pride

SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels.

Project description:Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.

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Driving Forces of Translocation Through Bacterial Translocon SecYEG.

Project description:This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG's conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.

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Specificity of SecYEG for PhoA precursors and SecA homologs on SecA protein-conducting channels.

Project description:Previous studies showed that Escherichia coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.

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Non-competitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in E. coli

Project description:The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro. However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD–SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is non-competitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.

2019-11-07 | PXD015985 | Pride

Noncompetitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in <i>Escherichia coli</i>.

Project description:The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.

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Binding of SecA ATPase monomers and dimers to lipid vesicles.

Project description:The Escherichia coli SecA ATPase motor protein is essential for secretion of proteins through the SecYEG translocon into the periplasmic space. Its function relies upon interactions with the surrounding lipid bilayer as well as SecYEG translocon. That negatively charged lipids are required for bilayer binding has been known for >25?years, but little systematic quantitative data is available. We have carried out an extensive investigation of SecA partitioning into large unilamellar vesicles (LUV) using a wide range of lipid and electrolyte compositions, including the principal cytoplasmic salt of E. coli, potassium glutamate, which we have shown stabilizes SecA. The water-to-bilayer transfer free energy is about -7.5?kcal?mol-1 for typical E. coli lipid compositions. Although it has been established that SecA is dimeric in the cytoplasm, we find that the most widely cited dimer form (PDB 1M6N) binds only weakly to LUVs formed from E. coli lipids.

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Charge-driven dynamics of nascent-chain movement through the SecYEG translocon.

Project description:On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.

| S-EPMC4338579 | biostudies-literature

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