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Donald Oliver - One of the best experts on this subject based on the ideXlab platform.
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the SecA protein deeply penetrates into the secyeg channel during insertion contacting most channel transmembrane helices and periplasmic regions
Journal of Biological Chemistry, 2017Co-Authors: Tithi Banerjee, Zeliang Zheng, Jane Abolafia, Shelby Harper, Donald OliverAbstract:Abstract The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific in vivo photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly-folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to “freeze” them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during in vitro protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane in order to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.
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SecA functions in vivo as a discrete anti parallel dimer to promote protein transport
Molecular Microbiology, 2017Co-Authors: Tithi Banerjee, Christine Lindenthal, Donald OliverAbstract:SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA x-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism. This article is protected by copyright. All rights reserved.
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conserved SecA signal peptide binding site revealed by engineered protein chimeras and forster resonance energy transfer
Biochemistry, 2016Co-Authors: Qi Zhang, Rich Olson, Ishita Mukerji, Donald OliverAbstract:Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In additio...
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dimeric SecA is essential for protein translocation
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Lucia B Jilaveanu, Christopher Zito, Donald OliverAbstract:SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal SecA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.
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role of a conserved glutamate residue in the escherichia coli SecA atpase mechanism
Journal of Biological Chemistry, 2005Co-Authors: Christopher R Zito, Donald Oliver, Edwin Antony, John F Hunt, Manju M HingoraniAbstract:Abstract Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis.
Phang C. Tai - One of the best experts on this subject based on the ideXlab platform.
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SecA inhibitors as potential antimicrobial agents differential actions on SecA only and SecA secyeg protein conducting channels
Fems Microbiology Letters, 2018Co-Authors: Jinshan Jin, Yinghsin Hsieh, Arpana S Chaudhary, Jianmei Cui, John E Houghton, Senfang Sui, Binghe Wang, Phang C. TaiAbstract: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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dissecting structures and functions of SecA only protein conducting channels atpase pore structure ion channel activity protein translocation and interaction with secyeg secdf yajc
PLOS ONE, 2017Co-Authors: Yinghsin Hsieh, John E Houghton, Senfang Sui, Hsiuchin Yang, Yingju Huang, Hao Zhang, Chun Jiang, Qian Liu, Phang C. TaiAbstract:SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the "Helical Scaffold" Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.
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phospholipids induce conformational changes of SecA to form membrane specific domains afm structures and implication on protein conducting channels
PLOS ONE, 2013Co-Authors: Zhipeng You, John E Houghton, Senfang Sui, Hsiuchin Yang, Meijiang Liao, Hao Zhang, Xijian Pan, Phang C. TaiAbstract:SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein-conducting channel in the membrane, without any requirement for SecYEG.
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Differential expression of secretion machinery during bacterial growth: SecY and SecF decrease while SecA increases during transition from exponential phase to stationary phase.
Current Microbiology, 2013Co-Authors: Chun-kai Yang, Phang C. TaiAbstract:Transcription of many house-keeping genes, including secY and some other sec genes, decreases in the transition from the exponential phase to the stationary phase (feast to famine) in Bacillus subtilis. Unexpectedly and in contradiction to earlier reports, enhanced transcription was observed for another group of sec genes, including SecA which codes for an essential ATPase for protein secretion. Consistent with the transcription data, the SecA protein of B. subtilis increases significantly in the stationary phase. Immunoblot analyses of Sec proteins during the transition in Escherichia coli also revealed the pronounced decreases of SecY and SecF and the increase of SecA, resulting in drastic increases of SecA/SecY and SecA/SecF ratios from exponential to stationary phases. The differential expression of Sec proteins in the stationary phase suggests the possibility of specific physiological functions.
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Phospholipids Induce Conformational Changes of SecA to Form Membrane-Specific Domains: AFM Structures and Implication on Protein-Conducting Channels
2013Co-Authors: Zhipeng You, John E Houghton, Hsiuchin Yang, Meijiang Liao, Hao Zhang, Xijian Pan, Phang C. TaiAbstract:SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein
Arnold J M Driessen - One of the best experts on this subject based on the ideXlab platform.
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Cellular dynamics of the SecA ATPase at the single molecule level
Scientific Reports, 2021Co-Authors: Anne-bart Seinen, Dian Spakman, Antoine M. Oijen, Arnold J M DriessenAbstract:In bacteria, the SecA ATPase provides the driving force for protein secretion via the SecYEG translocon. While the dynamic interplay between SecA and SecYEG in translocation is widely appreciated, it is not clear how SecA associates with the translocon in the crowded cellular environment. We use super-resolution microscopy to directly visualize the dynamics of SecA in Escherichia coli at the single-molecule level. We find that SecA is predominantly associated with and evenly distributed along the cytoplasmic membrane as a homodimer, with only a minor cytosolic fraction. SecA moves along the cell membrane as three distinct but interconvertible diffusional populations: (1) A state loosely associated with the membrane, (2) an integral membrane form, and (3) a temporarily immobile form. Disruption of the proton-motive-force, which is essential for protein secretion, re-localizes a significant portion of SecA to the cytoplasm and results in the transient location of SecA at specific locations at the membrane. The data support a model in which SecA diffuses along the membrane surface to gain access to the SecYEG translocon.
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covalently dimerized SecA is functional in protein translocation
Journal of Biological Chemistry, 2005Co-Authors: Jeanine De Keyzer, Eli O Van Der Sluis, Robin E J Spelbrink, Niels Nijstad, Ben De Kruijff, Nico Nouwen, Chris Van Der Does, Arnold J M DriessenAbstract:The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.
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the carboxyl terminus of the bacillus subtilis SecA is dispensable for protein secretion and viability
Biochemical Journal, 2000Co-Authors: Karel H M Van Wely, Roland Freudl, Michael Klein, Jelto Swaving, Arnold J M DriessenAbstract:The Escherichia coli secretion-dedicated chaperone SecB targets a subset of proteins to the translocase by interacting with the carboxyl (C-) terminus of SecA. This region of SecA is highly conserved in Eubacteria, but despite its presence in the Bacillus subtilis SecA, the B. subtilis genome does not appear to contain a gene for a clear homologue of SecB. Deletion of the C-terminus of the B. subtilis SecA yields cells that have normal viability, but that exhibit a response reminiscent of oxidative stress and the loss of a number of secretory proteins from the culture supernatant. Semi-quantitative RT-PCR demonstrates that these proteins are expressed at lower levels. The C-terminus of SecA fused to glutathione S-transferase (GST) specifically binds a cytosolic protein, termed MrgA. This protein has been reported to function in relation to oxidative stress, but deletion of the mrgA gene does not result in a secretion defect nor does it cause an oxidative stress response. It is concluded that the C-terminus of the B. subtilis SecA is not essential for secretion and viability.
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the molecular chaperone secb is released from the carboxy terminus of SecA during initiation of precursor protein translocation
The EMBO Journal, 1997Co-Authors: P Fekkes, Chris Van Der Does, Arnold J M DriessenAbstract:The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB–precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.
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identification of the magnesium binding domain of the high affinity atp binding site of the bacillus subtilis and escherichia coli SecA protein
Journal of Biological Chemistry, 1995Co-Authors: J P W Van Der Wolk, Roland Freudl, M Klose, De Janny Wit, Den T Blaauwen, Arnold J M DriessenAbstract:The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli SecA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.
Roland Freudl - One of the best experts on this subject based on the ideXlab platform.
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functional implementation of the posttranslational secb SecA protein targeting pathway in bacillus subtilis
Applied and Environmental Microbiology, 2012Co-Authors: Liuyang Diao, Jiahai Zhou, Qilei Dong, Sheng Yang, Roland FreudlAbstract:Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.
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the carboxyl terminus of the bacillus subtilis SecA is dispensable for protein secretion and viability
Biochemical Journal, 2000Co-Authors: Karel H M Van Wely, Roland Freudl, Michael Klein, Jelto Swaving, Arnold J M DriessenAbstract:The Escherichia coli secretion-dedicated chaperone SecB targets a subset of proteins to the translocase by interacting with the carboxyl (C-) terminus of SecA. This region of SecA is highly conserved in Eubacteria, but despite its presence in the Bacillus subtilis SecA, the B. subtilis genome does not appear to contain a gene for a clear homologue of SecB. Deletion of the C-terminus of the B. subtilis SecA yields cells that have normal viability, but that exhibit a response reminiscent of oxidative stress and the loss of a number of secretory proteins from the culture supernatant. Semi-quantitative RT-PCR demonstrates that these proteins are expressed at lower levels. The C-terminus of SecA fused to glutathione S-transferase (GST) specifically binds a cytosolic protein, termed MrgA. This protein has been reported to function in relation to oxidative stress, but deletion of the mrgA gene does not result in a secretion defect nor does it cause an oxidative stress response. It is concluded that the C-terminus of the B. subtilis SecA is not essential for secretion and viability.
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functional characterization of the staphylococcus carnosus SecA protein in escherichia coli and bacillus subtilis SecA mutant strains
Fems Microbiology Letters, 1995Co-Authors: Michael Klein, Jochen Meens, Roland FreudlAbstract:The Staphylococcus carnosus SecA gene was cloned using the Bacillus subtilis SecA gene as a probe. The S. carnosus SecA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B. subtilis SecA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli SecA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism.
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identification of the magnesium binding domain of the high affinity atp binding site of the bacillus subtilis and escherichia coli SecA protein
Journal of Biological Chemistry, 1995Co-Authors: J P W Van Der Wolk, Roland Freudl, M Klose, De Janny Wit, Den T Blaauwen, Arnold J M DriessenAbstract:The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli SecA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.
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characterization of a bacillus subtilis SecA mutant protein deficient in translocation atpase and release from the membrane
Molecular Microbiology, 1993Co-Authors: J P W Van Der Wolk, Roland Freudl, M Klose, Eefjan Breukink, R A Demel, B De Kruijff, Arnold J M DriessenAbstract:SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.
Tom A Rapoport - One of the best experts on this subject based on the ideXlab platform.
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protein translocation by the SecA atpase occurs by a power stroke mechanism
The EMBO Journal, 2019Co-Authors: Tom A Rapoport, Marco A Catipovic, Benedikt W Bauer, Joseph J LoparoAbstract:SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides.
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the bacterial atpase SecA functions as a monomer in protein translocation
Journal of Biological Chemistry, 2005Co-Authors: Dana Boyd, Stephanie Gon, Jonathan Beckwith, Tom A RapoportAbstract:The ATPase SecA drives the post-translational translocation of proteins through the SecY channel in the bacterial inner membrane. SecA is a dimer that can dissociate into monomers under certain conditions. To address the functional importance of the monomeric state, we generated an Escherichia coli SecA mutant that is almost completely monomeric (>99%), consistent with predictions from the crystal structure of Bacillus subtilis SecA. In vitro, the monomeric derivative retained significant activity in various assays, and in vivo, it sustained 85% of the growth rate of wild type cells and reduced the accumulation of precursor proteins in the cytoplasm. Disulfide cross-linking in intact cells showed that mutant SecA is monomeric and that even its parental dimeric form is dissociated. Our results suggest that SecA functions as a monomer during protein translocation in vivo.
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a large conformational change of the translocation atpase SecA
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andrew R Osborne, William M Clemons, Tom A RapoportAbstract:The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-A resolution. A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state.
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dissociation of the dimeric SecA atpase during protein translocation across the bacterial membrane
The EMBO Journal, 2002Co-Authors: Amiel Navon, Tom A RapoportAbstract:The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.
