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Results for “SEC Translocation Channels”

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PubMed1997

H+-pumping ATPase has little stimulatory effect on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles.

We studied the effect of a H+ electrochemical potential generated by F1F0 ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F1F0 ATPase of V. alginolyticus catalyzed the pumping of H+ coupled to ATP hydrolysis as measured in fluorescence quenching experiments. Consequently, this enzyme leads to the generation of a H+ electrochemical potential. The H+ electrochemical potential generated by F1F0 ATPase was completely abolished by 30 microM N,N'-dicyclohexylcarbodiimide (DCCD) or 5 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30 degrees C. The treatment of membrane vesicles with 30 microM DCCD at 30 degrees C had little influence on the translocation activity of uncleavable OmpF-Lpp, a model secretory protein, as compared to the intact membrane vesicles. On the other hand, the NADH:quinone oxidoreductase of V. alginolyticus is known to be a Na+ pump that leads to generation of a Na+ electrochemical potential. This Na+ electrochemical potential stimulates protein translocation into inside-out membrane vesicles prepared from V. alginolyticus in the presence of Escherichia coli SecA [Tokuda, H., Kim, Y. J., and Mizushima, S. (1990) FEBS Lett. 264, 10-12] From these results, it is evident that the stimulatory effect of the Na+ electrochemical potential on protein translocation in V. alginolyticus is not affected by the H+ electrochemical potential influence of F1F0 ATPase.

Adenosine Triphosphatases↗

PubMed1998

The role of molecular chaperones in protein transport into the mammalian endoplasmic reticulum.

Molecular chaperones belonging to the Hsp70 protein family play various roles in posttranslational protein transport into the yeast endoplasmic reticulum (ER): (i) Cytosolic Hsp70 (Hsc70) together with the J-domain containing Hsp40 preserves the transport competent state of presecretory proteins; (ii) ER-lumenal Hsp70 (Kar2p or Lhs1p) triggers the initial insertion of precursor proteins into the core of the protein translocase in the ER-membrane and cooperates with a J-domain containing membrane protein (Sec63p); (iii) the ER-lumenal Hsp70 and its membrane receptor also are involved in completion of translocation. Here a working model is presented for the putative roles of molecular chaperones in post- as well as cotranslational protein transport into the mammalian ER.

Animals↗

PubMed1998

The preprotein translocase of the mitochondrial outer membrane.

The translocase components of the mitochondrial outer membrane (Tom) are essential for the transfer of preproteins from the cytosol to the mitochondria. At least nine Tom components have now been identified and they fall into two categories; receptor components and components of the General Import Pore (GIP). The receptor components most likely diffuse laterally in the membrane and deposit preproteins atthe GIP where they are then inserted into the mitochondria. This review focuses on these components and their known interactions in the process of preprotein translocation.

Adenosine Triphosphatases↗

PubMed2000

Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum.

In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.

Chaperonin Containing TCP-1↗

PubMedDate not supplied

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review).

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.

Bacterial Proteins↗

PubMed2003

Secretins of Pseudomonas aeruginosa: large holes in the outer membrane.

Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.

Bacterial Outer Membrane Proteins↗

PubMed1999

Posttranslational protein translocation across the membrane of the endoplasmic reticulum.

Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.

Animals↗

PubMed1999

BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane.

We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.

Amino Acid Sequence↗

PubMed2001

Function of YidC for the insertion of M13 procoat protein in Escherichia coli: translocation of mutants that show differences in their membrane potential dependence and Sec requirement.

The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC. We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC. YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential. We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues. Furthermore, when Sec-dependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm. This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein. These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.

Adenosine Triphosphatases↗

PubMed2002

Three-dimensional structure of the bacterial protein-translocation complex SecYEG.

Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.

Cell Membrane↗

PubMed2006

In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering on one face of SecA.

The Sec-dependent protein translocation pathway promotes the transport of proteins into or across the bacterial plasma membrane. SecA ATPase has been shown to be a nanomotor that associates with its protein cargo as well as the SecYEG channel complex and to undergo ATP-driven cycles of membrane insertion and retraction that promote stepwise protein translocation. Previous studies have shown that both the 65-kDa N-domain and 30-kDa C-domain of SecA appear to undergo such membrane cycling. In the present study we performed in vivo sulfhydryl labeling of an extensive collection of monocysteine secA mutants under topologically specific conditions to identify regions of SecA that are accessible to the trans side of the membrane in its membrane-integrated state. Our results show that distinct regions of five of six SecA domains were labeled under these conditions, and such labeling clusters to a single face of the SecA structure. Our results demarcate an extensive face of SecA that interacts with SecYEG and is in fluid contact with the protein-conducting channel. The observed domain-specific labeling patterns should also provide important constraints on model building efforts in this dynamic system.

Adenosine Triphosphatases↗

PubMed2006

Selective photoaffinity labeling identifies the signal peptide binding domain on SecA.

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.

Adenosine Triphosphatases↗

PubMed1996

Sec-dependent preprotein translocation in bacteria.

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multisubunit protein complex termed translocase, which consists of the integral membrane heterotrimer Sec YEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with Sec YEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles.

Adenosine Triphosphatases↗

PubMed2004

SecA folding kinetics: a large dimeric protein rapidly forms multiple native states.

SecA, a 202 kDa dimeric protein, is the ATPase for the Sec-dependent translocase of precursor proteins in vivo. SecA must undergo conformational changes, which may involve dissociation into a monomer, as it translocates the precursor protein across the inner membrane. To better understand the dynamics of SecA in vivo, protein folding studies to probe the native, intermediate, and unfolded species of SecA in vitro have been done. SecA folds through a stable dimeric intermediate and dimerizes in the dead-time of a manual-mixing kinetic experiment ( approximately 5-7 seconds). Here, stopped-flow fluorescence and CD, as well as ultra-rapid continuous flow fluorescence techniques, were used to further probe the rapid folding kinetics of SecA. In the absence of urea, rapid, near diffusion-limited ( approximately 10(9)M(-1)s(-1)) SecA dimerization occurs following a rate-limiting unimolecular rearrangement of a rapidly formed intermediate. Multiple kinetic folding and unfolding phases were observed and SecA was shown to have multiple native and unfolded states. Using sequential-mixing stopped-flow experiments, SecA was determined to fold via parallel channels with sequential intermediates. These results confirm that SecA is a highly dynamic protein, consistent with the rapid, major conformational changes it must undergo in vivo.

Adenosine Triphosphatases↗

PubMed2001

SecG function and phospholipid metabolism in Escherichia coli.

SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of secG was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon. Introduction of both the glpR mutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains. It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.

Bacterial Proteins↗

PubMed1982

Dielectric relaxation studies of ionic processes in lysolecithin-packaged gramicidin channels.

Dielectric permitivities have been determined for suspensions of lysolecithin packaged malonyl gramicidin channels over the frequency range of 5 kHz to 900 MHz and under conditions of approximately equimolar concentrations (approximately 10mM) of channels and salts. The salts were lithium chloride, sodium chloride and thallium acetate. A relaxation process unique to the thallium acetate-channel system was observed which on analysis gave rise to a relaxation time at 25 degrees of 120 msec. The permitivity data, as well as a comparison of binding constants, indicate that the relaxation process results from TI+ being bound within the channel and more specifically from an intrachannel ion translocation with a rate constant of approximately 4 x 10(6) sec-1 and with an energy of activation of less than 6.7 kcal/mole. These data compare favorably with data from conductance studies on planar bilayers and with ion and carbon-13 nuclear magnetic studies on the lysolecithin packaged malonyl gramicidin channels which combine to indicate that the relaxation process is due to the jump of the thallium ion across a central barrier.

Circular Dichroism↗

PubMed1996

Polypeptide translocation machinery of the yeast endoplasmic reticulum.

Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. In Saccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of the Escherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK and S. cerevisiae Ssa1p, respectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of approximately 70 residue similarity to the 'J box', the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.

Cytoplasm↗

PubMed2006

Two gene determinants are differentially involved in the biogenesis of Fap1 precursors in Streptococcus parasanguis.

Mature Fap1, a 200-kDa fimbria-associated adhesin, is required for fimbrial biogenesis and biofilm formation in Streptococcus parasanguis. Fap1-like proteins are found in the genomes of many streptococcal and staphylococcal species. Fap1 is a serine-rich glycoprotein modified by O-linked glycan moieties. In this study, we identified a seven-gene cluster including secY2, orf1, orf2, orf3, secA2, gtf1, and gtf2 that is localized immediately downstream of fap1. The lower G+C contents and the presence of a putative transposase element suggest that this gene cluster was horizontally transferred from other bacteria and represents a genomic island. At least two genes in this island mediated Fap1 biogenesis. Mutation of a glucosyltransferase (Gtf1) gene led to accumulation of a Fap1 precursor, which had no detectable glycan moieties. Inactivation of a gene coding for an accessory Sec protein (SecY2) resulted in expression of a distinct Fap1 precursor, which reacted with one glycan-specific Fap1 antibody but not with another glycan-specific antibody. Furthermore, partially glycosylated Fap1 was detected on the cell surface and in the culture supernatant. These data suggest that SecY2 has a role in complete glycosylation of Fap1 and imply that SecY2 is not the only translocation channel for the Fap1 precursor and that alternative secretion machinery exists. Together, Gtf1 and SecY2 are involved in biogenesis of two distinct Fap1 precursors in S. parasanguis. Discovery of the effect of an accessory Sec protein on Fap1 glycosylation suggests that Fap1 secretion and glycosylation are coupled during Fap1 biogenesis.

Bacterial Proteins↗