42 results on '"secY"'
Search Results
2. Corrigendum: Dynamic nature of SecA and Its associated proteins in Escherichia coli
- Author
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Shun Adachi, Yasuhiro Murakawa, and Sota Hiraga
- Subjects
chromosome partition ,SecA ,SecY ,AcpP ,MukB ,DNA topoisomerase ,Microbiology ,QR1-502 - Published
- 2020
- Full Text
- View/download PDF
3. Molecular Mimicry of SecA and Signal Recognition Particle Binding to the Bacterial Ribosome
- Author
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Lara Knüpffer, Clara Fehrenbach, Kärt Denks, Veronika Erichsen, Narcis-Adrian Petriman, and Hans-Georg Koch
- Subjects
SecA ,SecY ,protein transport ,ribosomes ,signal recognition particle ,Microbiology ,QR1-502 - Abstract
ABSTRACT Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome. IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time.
- Published
- 2019
- Full Text
- View/download PDF
4. Two paths diverged in the stroma: targeting to dual SEC translocase systems in chloroplasts.
- Author
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Fernandez, Donna E.
- Abstract
Chloroplasts inherited systems and strategies for protein targeting, translocation, and integration from their cyanobacterial ancestor. Unlike cyanobacteria however, chloroplasts in green algae and plants contain two distinct SEC translocase/integrase systems: the SEC1 system in the thylakoid membrane and the SEC2 system in the inner envelope membrane. This review summarizes the mode of action of SEC translocases, identification of components of the SEC2 system, evolutionary history of SCY and SECA genes, and previous work on the co- and post-translational targeting of lumenal and thylakoid membrane proteins to the SEC1 system. Recent work identifying substrates for the SEC2 system and potential features that may contribute to inner envelope targeting are also discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
5. Uncovering the membrane-integrated SecAN protein that plays a key role in translocating nascent outer membrane proteins.
- Author
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Jin, Feng and Chang, Zengyi
- Subjects
- *
MEMBRANE proteins , *BACTERIAL proteins , *N-terminal residues , *BLOOD proteins , *PROTEINS , *AMINO acid residues - Abstract
A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecAN protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts. [Display omitted] • A hitherto unreported SecAN protein directly interacts with nascent OMPs. • SecAN seems to exist as a membrane-integrated homooligomer. • Precursor processing of OMPs is severely retarded when the SecAN assembly becomes defective due to mutations introduced in its GXXXG motif. • SecAN directly interacts with BamA in living cells. • SecAN shares the N-terminal 375 residues of SecA. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
6. Dynamic nature of SecA and its associated proteins in Escherichia coli.
- Author
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Shun eAdachi, Yasuhiro eMurakawa, and Sota eHiraga
- Subjects
secY ,DNA topoisomerase ,SecA ,acpP ,chromosome partition ,MukB ,Microbiology ,QR1-502 - Abstract
Mechanical properties such as physical constraint and pushing of chromosomes are thought to be important for chromosome segregation in Escherichia coli and it could be mediated by a hypothetical molecular tether. However, the actual tether that mediates these features is not known. We previously described that SecA (Secretory A) and SecY, components of the membrane protein translocation machinery, and AcpP (Acyl carrier protein P) were involved in chromosome segregation and homeostasis of DNA topology. In the present work we performed three-dimensional deconvolution of microscopic images and time-lapse experiments of these proteins together with MukB and DNA topoisomerases, and found that these proteins embraced the structures of tortuous nucleoids with condensed regions. Notably, SecA, SecY and AcpP dynamically localized in cells, which was interdependent on each other requiring the ATPase activity of SecA. Our findings imply that the membrane protein translocation machinery plays a role in the maintenance of proper chromosome partitioning, possibly through tethering of MukB (a functional homolog of structural maintenance of chromosomes [SMC] proteins), DNA gyrase, DNA topoisomerase IV and SeqA (Sequestration A).
- Published
- 2015
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- View/download PDF
7. Dynamic nature of SecA and Its associated proteins in Escherichia coli
- Author
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Sota Hiraga, Yasuhiro Murakawa, and Shun Adachi
- Subjects
Microbiology (medical) ,SecA ,biology ,Chemistry ,Topoisomerase ,lcsh:QR1-502 ,Correction ,SecY ,medicine.disease_cause ,Microbiology ,lcsh:Microbiology ,Biochemistry ,medicine ,biology.protein ,chromosome partition ,AcpP ,Escherichia coli ,MukB ,DNA topoisomerase - Published
- 2020
- Full Text
- View/download PDF
8. Corrigendum: Dynamic nature of SecA and Its associated proteins in Escherichia coli.
- Author
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Adachi, Shun, Murakawa, Yasuhiro, and Hiraga, Sota
- Subjects
ESCHERICHIA coli ,PROTEINS ,GREEN fluorescent protein ,MICROBIAL physiology ,THREE-dimensional imaging - Abstract
Keywords: chromosome partition; SecA; SecY; AcpP; MukB; DNA topoisomerase EN chromosome partition SecA SecY AcpP MukB DNA topoisomerase N.PAG N.PAG 2 01/18/21 20210113 NES 210113 In the original article, there were errors in Figures 1C,F and G as published. (B) SecA-GFP uv4, (C) SecY-GFP uv4, (D) AcpP-GFP uv4, (E) GFP uv4 (not fused with any protein), (F) No GFP uv4. Chromosome partition, SecA, SecY, AcpP, MukB, DNA topoisomerase. [Extracted from the article]
- Published
- 2021
- Full Text
- View/download PDF
9. Characterization of the supporting role of SecE in protein translocation.
- Author
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Lycklama a Nijeholt, Jelger A., de Keyzer, Jeanine, Prabudiansyah, Irfan, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *MOLECULAR chaperones , *PROTEIN stability , *BINDING sites , *PROTEIN-protein interactions , *CHROMOSOMES - Abstract
Highlights: [•] Sites of interaction between SecY and SecE remain immobile during channel opening. [•] The hinge region of SecE is essential for translocation. [•] SecE functions to guide and stabilize the open pore state of SecY. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
10. Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism
- Author
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Catipovic, Marco A, Bauer, Benedikt W, Loparo, Joseph J, and Rapoport, Tom A
- Published
- 2019
- Full Text
- View/download PDF
11. The bacterial Sec-translocase: structure and mechanism.
- Author
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Lycklama a Nijeholt, Jelger A. and Driessen, Arnold J. M.
- Subjects
- *
CELL membranes , *ADENOSINE triphosphate , *GRAM-negative bacteria , *MEMBRANE fusion , *ESCHERICHIA coli - Abstract
Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
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12. Structural determinants of protein translocation in bacteria: conformational flexibility of SecA IRA1 loop region.
- Author
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Palladino, Pasquale, Saviano, Gabriella, Tancredi, Teodorico, Benedetti, Ettore, Rossi, Filomena, and Ragone, Raffaele
- Subjects
PEPTIDES ,TYROSINE ,ESCHERICHIA coli ,PROTEIN conformation ,ENZYMES - Abstract
The article discusses a study which suggests a predominant flexible state for the synthetic wild-type peptide SecA and its analog SecA[788-804]Y794A except for peptides corresponding to SecA helices. It notes that the high flexibility of the SecA domain is independent of the presence of the functional loop tyrosine but can be fundamental SecA loop docking in SecY cavity and subsequent complex activation. The peptides E. coli were synthesized in groups. It suggests that the conformational flexibility of the peptides is not affected by Y794A substitution.
- Published
- 2011
- Full Text
- View/download PDF
13. Protein transport across the endoplasmic reticulum membrane.
- Author
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Rapoport, Tom A.
- Subjects
- *
PROTEINS , *PROKARYOTES , *ENDOPLASMIC reticulum , *CHROMOSOMAL translocation , *CELL membranes - Abstract
A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
14. Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—Distinct translocases and mechanisms
- Author
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Natale, Paolo, Brüser, Thomas, and Driessen, Arnold J.M.
- Subjects
- *
BIOLOGICAL transport , *CELL membranes , *FUNGUS-bacterium relationships , *SURFACE chemistry - Abstract
Abstract: In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
15. Protein Translocation Across the Bacterial Cytoplasmic Membrane.
- Author
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Driessen, Arnold J. M. and Nouwen, Nico
- Subjects
- *
PROTEINS , *CELL membranes , *BACTERIA , *MOLECULAR chaperones , *PROTONS - Abstract
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
16. Kinetics and Energetics of the Translocation of Maltose Binding Protein Folding Mutants
- Author
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Tomkiewicz, Danuta, Nouwen, Nico, and Driessen, Arnold J.M.
- Subjects
- *
CARRIER proteins , *PROTEIN folding , *ESCHERICHIA , *PROTEIN conformation - Abstract
Abstract: Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
17. Bacterial Sec-translocase Unfolds and Translocates a Class of Folded Protein Domains
- Author
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Nouwen, Nico, Berrelkamp, Greetje, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *BACTERIAL immunoglobulin-binding proteins , *MICROBIAL peptides , *BACTERIAL proteins - Abstract
Abstract: It is generally assumed that preprotein substrates must be presented in an unfolded state to the bacterial Sec-translocase in order to be translocated. Here, we have examined the ability of the Sec-translocase to translocate folded preproteins. Tightly folded human cardiac Ig-like domain I27 fused to the C terminus of proOmpA is translocated efficiently by the Sec-translocase and the translocation kinetics are determined by the extent of folding of the titin I27 domain. Accumulation of specific translocation intermediates around the fusion point that undergo translocation progress upon ATP binding suggests that the motor protein SecA plays an important and decisive role in promoting unfolding of the titin I27 domain. It is concluded that the bacterial Sec-translocase is capable of actively unfolding preproteins. [Copyright &y& Elsevier]
- Published
- 2007
- Full Text
- View/download PDF
18. Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation
- Author
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de Keyzer, Jeanine, Regeling, Anouk, and Driessen, Arnold J.M.
- Subjects
- *
ARGININE , *CHROMOSOMAL translocation , *ADENOSINE triphosphatase , *MEMBRANE proteins - Abstract
Abstract: The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only. [Copyright &y& Elsevier]
- Published
- 2007
- Full Text
- View/download PDF
19. Corrigendum: Dynamic nature of SecA and Its associated proteins in Escherichia coli.
- Author
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Adachi, Shun, Murakawa, Yasuhiro, and Hiraga, Sota
- Subjects
ESCHERICHIA coli ,DNA topoisomerase I ,PROTEINS - Abstract
Keywords: chromosome partition; SecA; SecY; AcpP; MukB; DNA topoisomerase EN chromosome partition SecA SecY AcpP MukB DNA topoisomerase N.PAG N.PAG 2 11/30/20 20201125 NES 201125 In the original article, there was a mistake in Figure 2 as published. (A) SecA-GFP uv4 in wild-type cells (MQ318) growing at 30°C. (E) SecA-GFP uv4 in acpP D38V(Ts) mutant cells (MQ456) growing at the non-permissive temperature of 42°C. [Extracted from the article]
- Published
- 2020
- Full Text
- View/download PDF
20. Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA
- Author
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van der Sluis, Eli O., Nouwen, Nico, Koch, Joachim, de Keyzer, Jeanine, van der Does, Chris, Tampé, Robert, and Driessen, Arnold J.M.
- Subjects
- *
GLUTATHIONE , *SERUM albumin , *BLOOD proteins , *CELL membranes - Abstract
Abstract: 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. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
- View/download PDF
21. The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity.
- Author
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de Keyzer, Jeanine, van der Does, Chris, Swaving, Jelto, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *PROTEIN-protein interactions , *SIGNAL peptides , *BILAYER lipid membranes , *BINDING sites , *SUPPRESSOR mutation , *HUMAN phenotype - Abstract
SecYEG forms the protein-conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity. [ABSTRACT FROM AUTHOR]
- Published
- 2002
- Full Text
- View/download PDF
22. SecYEG assembles into a tetramer to form the active protein translocation channel.
- Author
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Manting, Erik H., van der Does, Chris, Remigy, Hervé, Engel, Andreas, and Driessen, Arnold J. M.
- Subjects
- *
ESCHERICHIA coli , *CELL membranes , *BACTERIAL cell walls , *TETRAMERS (Oligomers) - Abstract
Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an ∼5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocaseprecursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes. [ABSTRACT FROM AUTHOR]
- Published
- 2000
- Full Text
- View/download PDF
23. Characterization of a Mutant Form of SecA That Alleviates a SecY Defect at Low Temperature and Shows a Synthetic Defect with SecY Alteration at High Temperature1.
- Author
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Nakatogawa, Hitoshi, Mori, Hiroyuki, Matsumoto, Gen, and Ito, Koreaki
- Subjects
MUTANT proteins ,PROTEIN binding ,TRYPSIN inhibitors ,TEMPERATURE ,HYDROLYSIS - Abstract
The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42°C. The overproduced SecA81 aggregated in vivo at temperatures above 37°C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30°C, while it was totally inactive at 42°C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421–575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42°C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42°C. The inactivated SecA81 molecules seemed to jam the translocate since it interfered with translocase functions at 42°C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY. [ABSTRACT FROM AUTHOR]
- Published
- 2000
- Full Text
- View/download PDF
24. Yet another job for the bacterial ribosome
- Author
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Origi, Andrea, Natriashivili, Ana, Knüpffer, Lara, Fehrenbach, Clara, Denks, Kärt, Asti, Rosella, and Koch, Hans-Georg
- Subjects
Models, Molecular ,Ribosomal Proteins ,SecA ,Molecular Biology and Physiology ,SecYEG ,uL23 ,Ribosome ,Binding, Competitive ,environment and public health ,Microbiology ,ribosomes ,03 medical and health sciences ,0302 clinical medicine ,Ribosomal protein ,Virology ,protein targeting ,Escherichia coli ,signal recognition particle ,030304 developmental biology ,0303 health sciences ,Signal recognition particle ,SecYEG Translocon ,Binding Sites ,SecA Proteins ,Chemistry ,Escherichia coli Proteins ,Cell Membrane ,Molecular Mimicry ,SecY ,Translation (biology) ,Microreview ,QR1-502 ,Transport protein ,Secretory protein ,ribosome ,Protein Biosynthesis ,Mutation ,Biophysics ,bacteria ,protein transport ,Signal recognition particle binding ,030217 neurology & neurosurgery ,Protein Binding ,Research Article - Abstract
Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time., Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome.
- Published
- 2019
25. PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.
- Author
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van der Wolk, Jeroen P. W., Fekkes, Peter, Boorsma, Andre, Huie, Janet L., Silhavy, Thomas J., and Driessen, Arnold J. M.
- Subjects
- *
ESCHERICHIA coli , *CELL membranes , *CHROMOSOMAL translocation , *ENZYMES , *GENETIC mutation , *PROTEIN precursors - Abstract
In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate · 8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, · 8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex. [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
- View/download PDF
26. The Escherichia coli SRP and SecB targeting pathways converge at the translocon.
- Author
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Valent, Quido A., Scotti, Pier A., High, Stephen, De Gier, Jan-Willem L., Von Heijne, Gunnar, Lentzen, Georg, Wintermeyer, Wolfgang, Oudega, Bauke, and Luirink, Joen
- Subjects
- *
ESCHERICHIA coli , *PROTEINS , *CELL membranes , *CHROMOSOMAL translocation - Abstract
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 preprotein 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. [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
- View/download PDF
27. Phylogeny and expression of the secA gene from a chromophytic alga – implications for the evolution of plastids and sec-dependent protein translocation.
- Author
-
Valentin, Klaus
- Abstract
In bacteria many periplasmatic proteins are exported via the sec-dependent pathway. A homologous apparatus was found to be involved in the transport of proteins across the thylakoid membrane in plastids. In the present study additional data on the phylogeny and expression of one of the genes essential in this process, secA, is presented. For the first time, transcriptional activity of secA in the plastid was detected. When secA is used as a phylogenetic marker for plastid evolution it demonstrates a large phylogenetic distance between chlorophytic and non-chlorophytic (i.e. rhodophytic) primary plastids. This distance could be explained by assuming polyphyly for major plastid lineages. Moreover, it was found that two types of secA genes may exist in plastids. Whether or not these are involved in different protein translocation processes is presently unknown. In an attempt to identify further candidates, i.e. non-photosynthesis-related proteins, for sec-dependent protein transport, an SbpA protein was detected in chromophytic plastids by the use of a peptide antibody. [ABSTRACT FROM AUTHOR]
- Published
- 1997
- Full Text
- View/download PDF
28. A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny.
- Author
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Vogel, Heiko, Fischer, Sabine, and Valentin, Klaus
- Abstract
Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen. We have analyzed one of the genes essential for this process, secY. A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid. Unlike the situation seen in bacteria the C. caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons. A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet. In this analysis plastid secY genes fall into two classes. One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart. Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga. All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues. We postulate that the two classes of secY genes are paralogous, i.e. their gene products are involved in different protein translocation processes. Based on this assumption a model for the evolution of the plastid sec apparatus is presented. [ABSTRACT FROM AUTHOR]
- Published
- 1996
- Full Text
- View/download PDF
29. The bacterial Sec-translocase: structure and mechanism
- Author
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Jelger A. Lycklama a Nijeholt and Arnold J. M. Driessen
- Subjects
Models, Molecular ,SecA ,Cytoplasm ,ENDOPLASMIC-RETICULUM ,Chromosomal translocation ,membrane protein insertion ,General Biochemistry, Genetics and Molecular Biology ,Motor protein ,Structure-Activity Relationship ,ESCHERICHIA-COLI TRANSLOCASE ,Adenosine Triphosphate ,Bacterial Proteins ,BACILLUS-SUBTILIS SECA ,Escherichia coli ,LARGE CONFORMATIONAL-CHANGE ,Translocase ,PRECURSOR PROTEIN TRANSLOCATION ,PLUG DOMAIN ,Bacterial Secretion Systems ,translocon ,Adenosine Triphosphatases ,SecA Proteins ,protein translocation ,ATP-BINDING-SITE ,biology ,Escherichia coli Proteins ,Cell Membrane ,SIGNAL-SEQUENCE RECOGNITION ,Membrane Transport Proteins ,SecY ,Articles ,Cell biology ,Enzyme Activation ,Protein Transport ,Membrane ,Secretory protein ,Biochemistry ,Membrane protein complex ,biology.protein ,PREPROTEIN TRANSLOCASE ,General Agricultural and Biological Sciences ,SEC Translocation Channels ,X-RAY-STRUCTURE ,Protein Binding - Abstract
Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.
- Published
- 2012
- Full Text
- View/download PDF
30. Protein Translocation Across the Bacterial Cytoplasmic Membrane
- Author
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Nico Nouwen and Arnold J. M. Driessen
- Subjects
SecA ,Cytoplasm ,Translocase of the outer membrane ,Molecular Conformation ,SIGNAL-RECOGNITION PARTICLE ,Models, Biological ,Biochemistry ,translocase ,PREPROTEIN TRANSLOCATION ,Twin-arginine translocation pathway ,Bacterial Proteins ,Escherichia coli ,chaperone ,Translocase ,membrane protein ,DISTINCT ATP-BINDING ,CRYSTAL-STRUCTURE ,ESCHERICHIA-COLI SECA ,STOP-TRANSFER FUNCTION ,Integral membrane protein ,Adenosine Triphosphatases ,SecA Proteins ,Bacteria ,biology ,PRECURSOR PROTEIN ,Escherichia coli Proteins ,proton motive force ,Cell Membrane ,Peripheral membrane protein ,SecY ,Membrane Transport Proteins ,Proton-Motive Force ,Protein Structure, Tertiary ,Cell biology ,Protein Transport ,Membrane protein complex ,Translocase of the inner membrane ,biology.protein ,Protons ,Peptides ,EXPORT CHAPERONE SECB ,SEC Translocation Channels ,X-RAY-STRUCTURE ,Molecular Chaperones - Abstract
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
- Published
- 2008
- Full Text
- View/download PDF
31. Kinetics and energetics of the translocation of maltose binding protein folding mutants
- Author
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Tomikiewicz, Danuta, Nouwen, Nico, Driessen, Arnold J. M., Tomkiewicz, Danuta, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials, and Molecular Microbiology
- Subjects
folding ,SecA ,PRECURSOR PROTEINS ,Protein Folding ,CATALYTIC CYCLE ,Chromosomal translocation ,Protein Structure, Secondary ,Maltose-binding protein ,LEADER SEQUENCE ,Structural Biology ,Translocase ,SIGNAL SEQUENCE ,Adenosine Triphosphatases ,protein translocation ,biology ,Escherichia coli Proteins ,TRIGGER FACTOR ,Tryptophan ,TERTIARY STRUCTURE ,SecY ,Folding (chemistry) ,Protein Transport ,Biochemistry ,ESCHERICHIA-COLI ,Periplasmic Binding Proteins ,Thermodynamics ,maltose binding protein ,Endopeptidase K ,Signal peptide ,Motor protein ,Bacterial Proteins ,Escherichia coli ,Protein Precursors ,Molecular Biology ,SecA Proteins ,COLI MEMBRANE-VESICLES ,Membrane Transport Proteins ,Protein tertiary structure ,Protein Structure, Tertiary ,Kinetics ,Spectrometry, Fluorescence ,Secretory protein ,Mutagenesis ,Mutation ,PLASMA-MEMBRANE ,biology.protein ,Biophysics ,CYTOPLASMIC MEMBRANE ,Mutant Proteins ,Carrier Proteins ,SEC Translocation Channels - Abstract
Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. (c) 2008 Elsevier Ltd. All rights reserved.
- Published
- 2008
32. Protein translocation across the bacterial cytoplasmic membrane
- Subjects
SecA ,PRECURSOR PROTEIN ,proton motive force ,SecY ,SIGNAL-RECOGNITION PARTICLE ,translocase ,PREPROTEIN TRANSLOCATION ,PROTON MOTIVE FORCE ,chaperone ,membrane protein ,DISTINCT ATP-BINDING ,CRYSTAL-STRUCTURE ,ESCHERICHIA-COLI SECA ,STOP-TRANSFER FUNCTION ,EXPORT CHAPERONE SECB ,X-RAY-STRUCTURE - Abstract
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they arc targeted to the See translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The See translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
- Published
- 2008
- Full Text
- View/download PDF
33. Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation
- Author
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Arnold J. M. Driessen, Anouk Regeling, Jeanine de Keyzer, and Molecular Microbiology
- Subjects
SecA ,CROSS-LINKING ,ATPase ,RIBOSOME-BINDING ,Biophysics ,INSERTION ,SIGNAL-RECOGNITION PARTICLE ,Chromosomal translocation ,Arginine ,Biochemistry ,Bacterial Proteins ,Structural Biology ,Genetics ,membrane insertion ,Protein Precursors ,Molecular Biology ,Adenosine Triphosphatases ,Signal recognition particle ,ARCHITECTURE ,SecA Proteins ,protein translocation ,COLI MEMBRANE-VESICLES ,COMPLEX ,biology ,IDENTIFICATION ,Escherichia coli Proteins ,Cell Membrane ,Membrane Proteins ,Membrane Transport Proteins ,SecY ,Cell Biology ,Periplasmic space ,Transmembrane protein ,Protein Transport ,Transmembrane domain ,Secretory protein ,Amino Acid Substitution ,Membrane protein ,Mutation ,biology.protein ,bacteria ,STEPS ,PROTEIN-CONDUCTING CHANNEL ,SEC Translocation Channels - Abstract
The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for 8357 in membrane protein insertion. Although 8357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that 8357 is essential for the initiation of SecA-dependent translocation only. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
- Published
- 2007
- Full Text
- View/download PDF
34. Identification of two interaction sites in SecY that are important for the functional interaction with SecA
- Author
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Chris van der Does, Eli O. van der Sluis, Robert Tampé, Jeanine de Keyzer, Joachim Koch, Nico Nouwen, Arnold J. M. Driessen, Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, and Faculty of Science and Engineering
- Subjects
SecA ,Cytoplasm ,CROSS-LINKING ,Glutamine ,environment and public health ,Ribosome ,Protein Structure, Secondary ,Structural Biology ,Membrane region ,CRYSTAL-STRUCTURE ,RIBOSOME ,TRANSLOCATION CHANNEL ,Adenosine Triphosphatases ,Signal recognition particle ,protein translocation ,Escherichia coli Proteins ,SecY ,Translocon ,Transmembrane domain ,Biochemistry ,MEMBRANE TRANSLOCATION ,peptide scanning ,PROTEIN-CONDUCTING CHANNEL ,Protein Binding ,SIGNAL RECOGNITION PARTICLE ,Molecular Sequence Data ,Biology ,Arginine ,Motor protein ,Structure-Activity Relationship ,Bacterial Proteins ,Escherichia coli ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,Binding Sites ,SecA Proteins ,ESCHERICHIA-COLI SECY ,Mutagenesis ,Membrane Transport Proteins ,cysteine crosslinking ,Kinetics ,BACTERIAL PROTEIN ,Biophysics ,bacteria ,PREPROTEIN TRANSLOCASE ,Peptides ,SEC Translocation Channels - Abstract
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. (c) 2006 Elsevier Ltd. All rights reserved.
- Published
- 2006
35. Dynamic hydrogen-bond networks in bacterial protein secretion.
- Author
-
Karathanou, Konstantina and Bondar, Ana-Nicoleta
- Subjects
- *
BACTERIAL proteins , *HYDROGEN bonding , *CRYSTAL structure - Abstract
The Sec protein secretion machinery includes proteins whose reaction coordinates involve large-scale conformational changes. Dynamic hydrogen-bond networks can provide structural plasticity required by the SecA protein motor and the SecY protein translocon. Here we discuss hydrogen-bond networks of these two Sec proteins from crystal structures and molecular simulations, and the usefulness of molecular simulations approaches to studying dynamic hydrogen bonds and their role in bacterial protein secretion. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
36. Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force
- Subjects
SECA ,MOTIVE FORCE ,PRO-OMPA ,SECE ,TRIGGER FACTOR ,PROTONMOTIVE FORCE ,CYTOCHROME-C OXIDASE ,SECY ,MALTOSE-BINDING PROTEIN ,EXPORT ,SECRETORY PROTEIN ,SECA PROTEIN ,INNER MEMBRANE-VESICLES ,PLASMA-MEMBRANE ,SECRETION - Abstract
The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (DELTA-p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a DELTA-p, inside acid and positive. DELTA-p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. DELTA-psi and DELTA-pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed DELTA-p. These results support a model [Schiebel,E., Driessen, A.J.M., Hartl,F.U. and Wickner,W. (1991) Cell, 64, 927-939] in which the DELTA-p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the DELTA-p challenges models involving DELTA-p-dependent membrane destabilization and provides further evidence for a role of the DELTA-p as driving force in precursor protein translocation.
- Published
- 1992
37. Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force
- Author
-
Arnold J.M. Driessen and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Delta ,SECA ,MOTIVE FORCE ,SECE ,PROTONMOTIVE FORCE ,Chromosomal translocation ,Diaphragm pump ,General Biochemistry, Genetics and Molecular Biology ,SECRETORY PROTEIN ,Maltose-binding protein ,Adenosine Triphosphate ,Methionine ,SECA PROTEIN ,Bacterial Proteins ,ATP hydrolysis ,Escherichia coli ,Translocase ,Cytochrome c oxidase ,Protein Precursors ,Molecular Biology ,Adenosine Triphosphatases ,SecA Proteins ,General Immunology and Microbiology ,biology ,PRO-OMPA ,Escherichia coli Proteins ,Hydrolysis ,TRIGGER FACTOR ,General Neuroscience ,CYTOCHROME-C OXIDASE ,Membrane Proteins ,Membrane Transport Proteins ,Biological Transport ,SECY ,Bacteriorhodopsin ,Hydrogen-Ion Concentration ,MALTOSE-BINDING PROTEIN ,EXPORT ,Biochemistry ,INNER MEMBRANE-VESICLES ,PLASMA-MEMBRANE ,biology.protein ,Biophysics ,SECRETION ,Protons ,SEC Translocation Channels ,Bacterial Outer Membrane Proteins ,Research Article - Abstract
The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (delta p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive. delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. delta psi and delta pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p. These results support a model [Schiebel, E., Driessen, A.J.M., Hartl, F.-U. and Wickner, W. (1991) Cell, 64, 927-939] in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving force in precursor protein translocation.
- Published
- 1992
- Full Text
- View/download PDF
38. Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms
- Author
-
Thomas Brüser, Paolo Natale, and Arnold J. M. Driessen
- Subjects
Signal peptide ,SecA ,Models, Molecular ,Protein Folding ,Biophysics ,Chromosomal translocation ,Biology ,Protein Sorting Signals ,Biochemistry ,TWIN-ARGININE TRANSLOCASE ,Models, Biological ,Twin-arginine translocation pathway ,03 medical and health sciences ,Adenosine Triphosphate ,Bacterial Proteins ,PROTON MOTIVE FORCE ,BACILLUS-SUBTILIS SECA ,Secretion ,ESCHERICHIA-COLI SECA ,030304 developmental biology ,Adenosine Triphosphatases ,0303 health sciences ,AMINO-TERMINAL REGION ,SecA Proteins ,ATP-BINDING-SITE ,030306 microbiology ,Twin arginine ,Escherichia coli Proteins ,Cell Membrane ,RIESKE FE/S PROTEIN ,SecY ,Membrane Transport Proteins ,Cell Biology ,Transmembrane protein ,Cell biology ,Protein Transport ,Secretory protein ,Protein folding ,Tat ,Carrier Proteins ,Energy Metabolism ,SEC Translocation Channels ,EXPORT CHAPERONE SECB ,THYLAKOID LUMEN PROTEIN ,X-RAY-STRUCTURE ,Metabolic Networks and Pathways - Abstract
In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrame translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different. (C) 2007 Elsevier B.V. All rights reserved.
- Published
- 2007
39. Bacterial sec-translocase unfolds and translocates a class of folded protein domains
- Author
-
Greetje Berrelkamp, Nico Nouwen, Arnold J. M. Driessen, and Molecular Microbiology
- Subjects
folding ,SecA ,Models, Molecular ,Protein Denaturation ,Protein Folding ,Protein domain ,Immunoglobulins ,Muscle Proteins ,Motor protein ,LEADER SEQUENCE ,Protein structure ,Adenosine Triphosphate ,Bacterial Proteins ,Structural Biology ,Escherichia coli ,Humans ,Urea ,Connectin ,Molecular Biology ,Adenosine Triphosphatases ,protein translocation ,SecA Proteins ,biology ,C-terminus ,IMPORT ,SecY ,Membrane Transport Proteins ,MALTOSE-BINDING PROTEIN ,EXPORT ,Transport protein ,Protein Structure, Tertiary ,ATP ,Protein Transport ,Biochemistry ,ESCHERICHIA-COLI ,MECHANICAL STABILITY ,biology.protein ,Biophysics ,PREPROTEIN TRANSLOCASE ,Titin ,Protein folding ,DIHYDROFOLATE-REDUCTASE ,SEC Translocation Channels ,Protein Kinases ,MEMBRANE-VESICLES ,Bacterial Outer Membrane Proteins - Abstract
It is generally assumed that preprotein substrates must be presented in an unfolded state to the bacterial Sec-translocase in order to be translocated. Here, we have examined the ability of the Sec-translocase to translocate folded preproteins. Tightly folded human cardiac Ig-like domain 12 7 fused to the C terminus of proOmpA is translocated efficiently by the Sectranslocase and the translocation kinetics are determined by the extent of folding of the titin 127 domain. Accumulation of specific translocation intermediates around the fusion point that undergo translocation progress upon ATP binding suggests that the motor protein SecA plays an important and decisive role in promoting unfolding of the titin 127 domain. It is concluded that the bacterial Sec-translocase is capable of actively unfolding preproteins. (C) 2007 Elsevier Ltd. All rights reserved.
- Published
- 2007
40. PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA–SecY interaction during the initiation of translocation
- Author
-
Janet L. Huie, P. Fekkes, André Boorsma, Arnold J. M. Driessen, Jeroen P.W. van der Wolk, Thomas J. Silhavy, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Signal peptide ,SecA ,Mutant ,Chromosomal translocation ,Protein Sorting Signals ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Bacterial Proteins ,Translocase ,Protein Precursors ,Molecular Biology ,Adenosine Triphosphatases ,SecYEG Translocon ,SecA Proteins ,General Immunology and Microbiology ,biology ,Membrane transport protein ,Escherichia coli Proteins ,General Neuroscience ,Membrane Transport Proteins ,SecY ,Biological Transport ,Cell biology ,Biochemistry ,Cytoplasm ,biology.protein ,signal sequence ,proof-reading ,bacteria ,SEC Translocation Channels ,Research Article ,Bacterial Outer Membrane Proteins - Abstract
In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.
- Published
- 1998
41. The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity
- Author
-
Jeanine de Keyzer, Arnold J. M. Driessen, Jelto Swaving, Chris van der Does, Moleculaire Microbiologie, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Signal peptide ,SecA ,ATPase ,Biophysics ,Chromosomal translocation ,Protein Sorting Signals ,medicine.disease_cause ,Biochemistry ,environment and public health ,Heating ,Bacterial Proteins ,Structural Biology ,Genetics ,medicine ,Translocase ,Signal sequence suppression ,GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries) ,Molecular Biology ,Escherichia coli ,Adenosine Triphosphatases ,Mutation ,Protein translocation ,SecA Proteins ,biology ,Membrane transport protein ,Escherichia coli Proteins ,Membrane Transport Proteins ,SecY ,Biological Transport ,Cell Biology ,Cell biology ,Phenotype ,Mutagenesis, Site-Directed ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,biology.protein ,bacteria ,SEC Translocation Channels - Abstract
SecYEG forms the protein-conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity.
- Full Text
- View/download PDF
42. Characterization of the supporting role of SecE in protein translocation
- Author
-
Jeanine de Keyzer, Jelger A. Lycklama a Nijeholt, Irfan Prabudiansyah, Arnold J. M. Driessen, Molecular Microbiology, and Enzymology
- Subjects
SecA ,CROSS-LINKING ,SecE ,Archaeal Proteins ,Amino Acid Motifs ,Biophysics ,Protein Export ,Biology ,Cleavage (embryo) ,Biochemistry ,Protein Structure, Secondary ,Bacterial Proteins ,Structural Biology ,Escherichia coli ,Genetics ,Protein Interaction Domains and Motifs ,Disulfides ,Protein translocation ,Molecular Biology ,Adenosine Triphosphatases ,Membrane transport ,Binding Sites ,SecA Proteins ,Escherichia coli Proteins ,MEMBRANE COMPONENT ,Disulfide bond ,Membrane Transport Proteins ,SecY ,CONSERVED CYTOPLASMIC REGION ,Methanococcaceae ,Cell Biology ,Protein Transport ,Transmembrane domain ,ESCHERICHIA-COLI ,PREPROTEIN TRANSLOCASE ,MACHINERY ,Hinge region ,SEC Translocation Channels ,Protein Binding - Abstract
SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic a-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
- Full Text
- View/download PDF
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