The Experts below are selected from a list of 201 Experts worldwide ranked by ideXlab platform
Arnold J. M. Driessen - One of the best experts on this subject based on the ideXlab platform.
-
The Canonical and Accessory Sec System of Gram-positive Bacteria
Current Topics in Microbiology and Immunology, 2016Co-Authors: Irfan Prabudiansyah, Arnold J. M. DriessenAbstract:The Sec system is present in all bacteria and responsible for the translocation of the majority of Proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor Protein, SecA, and the Protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory Proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.
-
Characterization of the annular lipid shell of the Sec translocon.
Biochimica et biophysica acta, 2015Co-Authors: Irfan Prabudiansyah, Ilja Kusters, Antonella Caforio, Arnold J. M. DriessenAbstract:The bacterial Sec translocase in its minimal form consists of a membrane-embedded Protein-conducting pore SecYEG that interacts with the motor Protein SecA to mediate the translocation of secretory Proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane Protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.
-
In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis
PloS one, 2015Co-Authors: Irfan Prabudiansyah, Ilja Kusters, Arnold J. M. DriessenAbstract:The majority of Proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor Protein SecA that associates with the Protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping Protein, whereas SecA2 is not essential but is involved in the translocation of a subset of Proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 Protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY Protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent Proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both Proteins are present simultaneously, they form dissociable heterodimers.
-
Mechanistic insights on the preProtein translocase: a single molecule study (362.4)
The FASEB Journal, 2014Co-Authors: Arnold J. M. DriessenAbstract:The heterotrimeric SecYEG complex comprises a Protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor Protein SecA in preProtein translocation. SecYEG can also associate with a translating ribosome to mediate membrane Protein insertion. We have used single molecule techniques, fluorescence spectroscopy and advanced membrane Protein reconstitution methods to address mechanistic and structural questions on the Protein translocase with the challenge to visualize single Protein translocation events.
-
Single-Molecule Studies of Bacterial Protein Translocation
Biochemistry, 2013Co-Authors: Alexej Kedrov, Ilja Kusters, Arnold J. M. DriessenAbstract:In prokaryotes, a large share of the Proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane Proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the Protein-conducting channel (SecYEG) that associates with soluble components, such as the motor Protein SecA or translating ribosomes, and with integral membrane Proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of Protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of Protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process.
Debra A Kendall - One of the best experts on this subject based on the ideXlab platform.
-
The SecA Dimer Interface
Biophysical Journal, 2014Co-Authors: Andy J. Wowor, Debra A Kendall, Yuetian Yan, Sarah M. Auclair, Michael L. Gross, James L. ColeAbstract:As a central component in the general secretion (Sec) pathway of bacteria, the ATPase motor Protein, SecA, mediates preProtein translocation through the integral membrane channel, SecYEG. SecA is a potential target for antibacterial therapeutics because it is crucial for Protein transport and cell viability. It is highly conserved among species of bacteria, yet it has no close human homologs. In the Sec pathway, SecA interacts with various ligands, including other SecA molecules. The latter interaction, a monomer-dimer equilibrium, is highly sensitive to salt concentration, temperature, and ligand binding. Although the structure of the SecA protomer is well-conserved among bacterial homologs, multiple dimer interfaces have been identified in SecA dimer crystal structures. We employed several biophysical methods to define the SecA dimer interface in solution and map the dimer interface using strategies independent of those used to solve the crystal structures of SecA dimer. By measuring the effects of alanine substitution on dimerization energetics using sedimentation velocity analytical ultracentrifugation, we determined that the substitution of residues at the N-terminus and within the helical scaffold domain significantly decreases dimerization affinity. By monitoring the backbone hydrogen/deuterium exchange rates using mass spectrometry, we found that residues lying within the helical scaffold domain are protected from exchange in the dimer state, consistent with the analytical ultracentrifugation results. These data are consistent with the Bacillus subtilis (1M6N) and Thermus thermophilus (2IPC) SecA dimeric structures.
-
Defining the Escherichia coli SecA Dimer Interface Residues through In Vivo Site-Specific Photo-Cross-Linking
Journal of bacteriology, 2013Co-Authors: Andy J. Wowor, James L. Cole, Debra A KendallAbstract:ABSTRACT The motor Protein SecA is a core component of the bacterial general secretory (Sec) pathway and is essential for cell viability. Despite evidence showing that SecA exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the SecA dimer. Here, a site-directed photo-cross-linking technique was utilized to identify residues on the Escherichia coli SecA (ecSecA) dimer interface in the cytosol of intact cells. The feasibility of this method was demonstrated with residue Leu6, which is essential for ecSecA dimerization based on our analytical ultracentrifugation studies of SecA L6A and shown to form the cross-linked SecA dimer in vivo with p-benzoyl-phenylalanine (pBpa) substituted at position 6. Subsequently, the amino terminus (residues 2 to 11) in the nucleotide binding domain (NBD), Phe263 in the preProtein binding domain (PBD), and Tyr794 and Arg805 in the intramolecular regulator of the ATPase 1 domain (IRA1) were identified to be involved in ecSecA dimerization. Furthermore, the incorporation of pBpa at position 805 did not form a cross-linked dimer in the SecA Δ2-11 context, indicating the possibility that the amino terminus may directly contact Arg805 or that the deletion of residues 2 to 11 alters the topology of the naturally occurring ecSecA dimer.
-
Demonstration of a specific Escherichia coli SecY-signal peptide interaction.
Biochemistry, 2004Co-Authors: Ligong Wang, Sharyn L. Rusch, Alexander Miller, Debra A KendallAbstract:Protein translocation in Escherichia coli is initiated by the interaction of a preProtein with the membrane translocase composed of a motor Protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preProtein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preProtein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the Protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA.
Nico Nouwen - One of the best experts on this subject based on the ideXlab platform.
-
Probing the SecYEG translocation pore size with preProteins conjugated with sizable rigid spherical molecules
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Francesco Bonardi, Nico Nouwen, Erik Halza, Martin Walko, François Du Plessis, Ben L. Feringa, Arnold J. M. DriessenAbstract:Abstract Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the Protein-conducting channel SecYEG, and the motor Protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded Proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to . To access the functional size of the SecYEG pore, the precursor of outer membrane Protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane Protein A conjugates carrying tetraarylmethanes with diameters up to , whereas a sized molecule blocked the translocation pore. Translocation of the Protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of , the pore accommodates structures of at least , which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.
-
Protein translocation across the bacterial cytoplasmic membrane
Annual Review of Biochemistry, 2008Co-Authors: Arnold J. M. Driessen, Nico NouwenAbstract: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.
-
Kinetics and energetics of the translocation of maltose binding Protein folding mutants
Journal of molecular biology, 2008Co-Authors: Danuta Tomkiewicz, Nico Nouwen, Arnold J. M. DriessenAbstract: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.
-
bacterial sec translocase unfolds and translocates a class of folded Protein domains
Journal of Molecular Biology, 2007Co-Authors: Nico Nouwen, Greetje Berrelkamp, Arnold J. M. DriessenAbstract: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.
-
Identification of two interaction sites in SecY that are important for the functional interaction with SecA.
Journal of molecular biology, 2006Co-Authors: Eli O. Van Der Sluis, Nico Nouwen, Joachim Koch, Jeanine De Keyzer, Chris Van Der Does, Robert Tampé, Arnold J. M. DriessenAbstract: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.
Ilja Kusters - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of the annular lipid shell of the Sec translocon.
Biochimica et biophysica acta, 2015Co-Authors: Irfan Prabudiansyah, Ilja Kusters, Antonella Caforio, Arnold J. M. DriessenAbstract:The bacterial Sec translocase in its minimal form consists of a membrane-embedded Protein-conducting pore SecYEG that interacts with the motor Protein SecA to mediate the translocation of secretory Proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane Protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.
-
In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis
PloS one, 2015Co-Authors: Irfan Prabudiansyah, Ilja Kusters, Arnold J. M. DriessenAbstract:The majority of Proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor Protein SecA that associates with the Protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping Protein, whereas SecA2 is not essential but is involved in the translocation of a subset of Proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 Protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY Protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent Proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both Proteins are present simultaneously, they form dissociable heterodimers.
-
Single-Molecule Studies of Bacterial Protein Translocation
Biochemistry, 2013Co-Authors: Alexej Kedrov, Ilja Kusters, Arnold J. M. DriessenAbstract:In prokaryotes, a large share of the Proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane Proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the Protein-conducting channel (SecYEG) that associates with soluble components, such as the motor Protein SecA or translating ribosomes, and with integral membrane Proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of Protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of Protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process.
-
A single copy of SecYEG is sufficient for preProtein translocation
The EMBO journal, 2011Co-Authors: Alexej Kedrov, Ilja Kusters, Victor V. Krasnikov, Arnold J. M. DriessenAbstract:The heterotrimeric SecYEG complex comprises a Protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor Protein SecA in preProtein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preProtein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Forster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preProtein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preProtein translocation intermediate. Neither the interaction with SecA nor preProtein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.
-
Taming membranes: functional immobilization of biological membranes in hydrogels.
PloS one, 2011Co-Authors: Ilja Kusters, Sander J. Tans, Nobina Mukherjee, Menno R. De Jong, Armagan Kocer, Arnold J. M. DriessenAbstract:Single molecule studies on membrane Proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with Protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane Proteins immobilized in the hydrogel as well as soluble Proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, Protein translocation activity by the membrane embedded Protein conducting channel of bacteria, SecYEG, in association with the soluble motor Protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane Proteins in their native environment at the single molecule level by means of fluorescence microscopy.
Peter Pohl - One of the best experts on this subject based on the ideXlab platform.
-
Interaction of the motor Protein SecA and the bacterial Protein translocation channel SecYEG in the absence of ATP
Nanoscale Advances, 2020Co-Authors: Klemens Winkler, Andreas Karner, Andreas Horner, Christof Hannesschlaeger, Denis G. Knyazev, Christine Siligan, Mirjam Zimmermann, Roland Kuttner, Peter Pohl, Johannes PreinerAbstract:Translocation of many secretory Proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor Protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG–SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of Protein translocation.
-
Binding of the motor Protein SecA to the bacterial Protein translocation channel SecYEG in the absence of ATP
2019Co-Authors: Klemens Winkler, Andreas Karner, Andreas Horner, Christof Hannesschlaeger, Denis G. Knyazev, Christine Siligan, Mirjam Zimmermann, Roland Kuttner, Peter Pohl, Johannes PreinerAbstract:Translocation of many secretory Proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor Protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecAs two-helix finger into SecYEGs aqueous channel. Such movement of the finger is in line with the power stroke mechanism of Protein translocation.
-
Forces and Dynamics in Protein Translocation through the Bacterial Translocon
Biophysical Journal, 2017Co-Authors: Anny Fis, Andreas Karner, Mirjam Zimmermann, Roland Kuttner, Peter Pohl, Johannes Preiner, Hermann J. Gruber, Peter HinterdorferAbstract:Many membrane and secretory Proteins are translocated across the endoplasmic reticulum membrane or the bacterial/archaeal plasma membrane through a conserved channel, the Sec61 or the SecYEG complex, respectively. In post-translational translocation the SecYEG channel associates with the cytoplasmic motor Protein SecA to deliver secretory Proteins to the periplasm. In this study we characterized the binding of SecA to SecYEG at the single molecule level. For our AFM measurements, a sandwich structure was established that consisted of a mica-supported lipid bilayer containing biotinylated lipids, followed by a streptavidin layer, and SecYEG reconstituted into planar lipid bilayers, also containing biotinylated lipids. By covalently coupling SecA to AFM cantilevers we performed combined recognition imaging and force spectroscopy experiments. By varying the loading rate we obtained information on interaction forces, energy landscapes and kinetic rate-constants of the bond formed between SecA and SecYEG. No significant differences were observed in the presence and absence of ATP. In contrast, SecA only binds in the presence of ATP to SecYEG in detergent. This observation suggests that ATP and the lipid bilayer might induce similar conformational changes in SecA. However, ATP did not restore binding of a N-terminal deletion mutant of SecA to either pure lipid bilayers or reconstituted SecYEG, raising the possibility that SecYEG¯s binding interface adopts different conformations in detergent and the lipid environment. This work is supported by the Austrian Science Fund (FWF), no.W1250-B200.
-
Robust Ligand Binding to the Protein Translocation Complex (Secyeg) Requires a Lipid Environment
Biophysical Journal, 2017Co-Authors: Klemens Winkler, Andreas Horner, Denis G. Knyazev, Christine Siligan, Roland Kuttner, Peter PohlAbstract:Many secretory Proteins pass the membrane barrier via the bacterial SecY complex. SecY is assisted by the motor Protein SecA, which is thought to push the polypeptides through the SecY pore with its two-helix finger. This model requires ATP molecules to exchange continuously during translocation. Whether the resulting change in SecA-SecY binding affinity leads to SecA release is unknown. Here we have tested the hypothesis by monitoring SecA binding to the SecY complex using lanthanide resonance energy transfer between a fluorescent label on the tip of its two-helix finger and Tb3+ in a genetically engineered binding pocket on SecY. In the absence of ATP, binding only occurred to SecY molecules that were reconstituted into lipid bilayers, but not to SecY molecules in detergent. In a fraction of these binding events we observed a deep insertion of SecA's two-helix finger into the SecY-channel. These penetrations resulted in relocation of SecY's plug as indicated by measurements of the distance between fingertip and plug. The ‘‘plug’’ is a short helix that is (i) normally located in the center of the channel adjacent to its hydrophobic constriction zone (pore ring) and (ii) blocks the passage of small molecules through the idle SecY complex (1). Observation of ion channel activity in reconstituted planar lipid bilayers confirmed that the plug had moved out of the pore. As previously observed in the case of ribosome nascent chain complexes (2), physiological values of the membrane potential were required to close the channel. We conclude that (i) SecA binding is sufficient to open the Protein translocation channel in its lipid environment and (ii) the current transport model needs to be revised to account for SecY's voltage sensitivity.The project was supported by the Austrian Science Fund (P25844, P28213).1. Saparov SM,..,Pohl P (2007) Mol Cell 26:501-509.2. Knyazev DG, ⋯, Pohl P (2014) J Biol Chem 289:24611-24616.
-
Voltage Sensitivity of the Bacterial Protein Translocation Channel
Biophysical Journal, 2016Co-Authors: Denis G. Knyazev, Christine Siligan, Roland Kuttner, Lukas Winter, Peter PohlAbstract:The heterotrimeric Protein translocation channel SecYEG enables (i) soluble Proteins to cross the inner membrane and (ii) hydrophobic Proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor Protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes. The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.1. Saparov, S.M., Erlandson, K., Cannon, K., Schaletzky, J., Schulman, S., Rapoport, T.A., Pohl, P. (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell.2. Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem.3. Knyazev, D.G., Winter, L., Bauer, B.W., Siligan, C., Pohl, P. (2014). Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation. J. Biol. Chem.
