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Results for “SEC Translocation Channels”

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PubMed2001

A marriage of bacteriology with cell biology results in twin arginines.

The Tat apparatus is a remarkable molecular machine required for the transmembrane translocation of folded proteins. Recent studies have identified jellyfish green fluorescent protein as an excellent reporter protein for the bacterial Tat pathway. Although the genetics of the Tat system are reasonably well-defined in Escherichia coli, the utilization of heterologous proteins as transport substrates promises to facilitate further mechanistic and structural characterization of this system.

Adenosine Triphosphatases↗

PubMed1999

Following the leader: bacterial protein export through the Sec pathway.

Significant strides have been made during the past 20 years in our understanding of protein secretion across the bacterial inner membrane. Specialized chaperones select secretory polypeptide chains and usher them to a membrane-embedded preprotein translocase. This unique molecular machine envelops the polymeric substrate and migrates along its length in defined, energy-dependent steps. Consequently, preproteins are gradually pumped into the periplasm where they acquire their native, folded conformation.

Adenosine Triphosphatases↗

PubMed2001

The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes.

In the evolution of mitochondria and plastids from endosymbiotic bacteria, most of the proteins that make up these organelles have become encoded by nuclear genes and must therefore be transported across the organellar membranes, following synthesis in the cytosol. The core component of the protein translocation machines in both the mitochondrial and plastid outer membranes appears to be a beta-barrel protein, perhaps a relic from their bacterial ancestry, distinguishing these translocases from the alpha-helical-based protein translocation pores found in all other eukaryotic membranes.

Bacterial Proteins↗

PubMed2002

Sec61beta--a component of the archaeal protein secretory system.

Sec61p/SecYEG complexes mediate protein translocation across membranes and are present in both eukaryotes and bacteria. Whereas homologues of Sec61alpha/SecY and Sec61gamma/SecE exist in archaea, identification of the third component (Sec61beta or SecG) has remained elusive. Using PSI-BLAST, the archaeal counterpart of Sec61beta has been detected. With the identification of the Sec61beta motif, functions for a universal family of archaeal proteins can be predicted and the archaeal translocon system can be definitively detected.

Amino Acid Motifs↗

PubMed1999

Cloning and nucleotide sequencing of the secA gene from coryneform bacteria.

Taking advantage of highly conserved domains present in the secA gene from Escherichia coli and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a lambda library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 5.3-kb EcoR1 fragment containing the secA homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum secA homolog is composed of 845 amino acids (aa) with a deduced molecular weight (MW) of 95429. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation and suggested that the Br. flavum secA homolog has putative ATP binding regions.

Adenosine Triphosphatases↗

PubMed1998

Regulation of protein topology by trans-acting factors at the endoplasmic reticulum.

In mammalian cells, the Sec61 complex and translocating chain-associated membrane protein (TRAM) are necessary and sufficient to direct the biogenesis, in the appropriate topology, of all secretory and membrane proteins examined thus far. We demonstrate here that the proper translocation of the prion protein (PrP), a substrate that can be synthesized in more than one topologic form, requires additional factors. In the absence of these additional factors, PrP is synthesized exclusively in the transmembrane topology (termed the CtmPrP form) associated with the development of neurodegenerative disease. Thus, translocation accessory factors, acting on some but not other substrates, can function as molecular switches to redirect nascent proteins toward divergent topologic fates with different functional consequences.

Animals↗

PubMed2001

Secretion monitor, SecM, undergoes self-translation arrest in the cytosol.

The product of the Escherichia coli secM gene (secretion monitor, formerly gene X), upstream of secA, is involved in secretion-responsive control of SecA translation. In wild-type cells, SecM is rapidly degraded by the periplasmic tail-specific protease. It is also subject to a transient translation pause at a position close to the C terminus. The elongation arrest was strikingly prolonged when translocation of SecM was impaired. SRP was not required for this arrest. Instead, the nascent SecM product itself may participate, as the arrest was diminished when it incorporated a proline analog, azetidine. We propose that cytosolically localized nascent SecM undergoes self-translation arrest, thereby enhancing translation of secA through an altered secondary structure of the secM-secA messenger RNA.

Adenosine Triphosphatases↗

PubMed2001

Protein transport: two translocons are better than one.

The translocon is the gateway to the endoplasmic reticulum (ER). In yeast this is the Sec61p complex. However, new evidence suggests that a second translocon containing the Sec61p homolog Ssh1p provides important flexibility to the ER translocation machinery.

Endoplasmic Reticulum↗

PubMed2003

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin.

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.

Animals↗

PubMed2003

Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins.

During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM alpha helix and signal-anchor sequence always faces Sec61alpha in the translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61alpha and is retained at a fixed location within the translocon, observations that are best explained by the binding of each TM sequence to a translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the translocon, nascent chain movement through the translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.

Bacterial Proteins↗

PubMed2000

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

Adenosine Triphosphatases↗

PubMed1998

The Sec system.

Proteins designated to be secreted by Escherichia coli are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB. Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecF. Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism or preprotein translocation. It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction.

Adenosine Triphosphatases↗

PubMed2001

Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum.

Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER). However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined. We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation. Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein. In addition, deltassh1 mutant cells rapidly became respiratory deficient. The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced. These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.

Animals↗

PubMed2000

SecA folds via a dimeric intermediate.

Though many proteins in the cell are large and multimeric, their folding has not been extensively studied. We have chosen SecA as a folding model because it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent preprotein translocase of many bacteria. SecA is a soluble protein that can penetrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functions in vivo. Kinetic folding transitions in the presence of urea were monitored using circular dichroism and tryptophan fluorescence, while equilibrium folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition exhibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N(2) if I(2) if 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. The SecA dimer was determined to have an overall stability (DeltaG) of -22.5 kcal/mol. We also investigated the stability of SecA using analytical ultracentrifugation equilibrium and velocity sedimentation, which again indicated that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the native dimer. Unfolding of native, dimeric SecA was slow with a relaxation time in H(2)O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.

Adenosine Triphosphatases↗

PubMed1993

Purified Escherichia coli preprotein translocase catalyzes multiple cycles of precursor protein translocation.

Escherichia coli preprotein translocase, composed of the peripheral membrane protein SecA bound at the integral membrane domain SecY/E, has been isolated and functionally reconstituted [Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., & Wickner, W. (1990) Cell 62, 649-657]. It is not known whether this purified enzyme supports multiple turnover cycles and how its kinetics compare with translocase in inverted membrane vesicles. We now report a quantitative comparison of the translocation of the outer membrane protein A precursor (proOmpA) by purified preprotein translocase and by inner membrane vesicles. ProOmpA cross-linked to bovine pancreatic trypsin inhibitor was used for quantitative titration of the functional translocation sites. The rate of proOmpA translocation per active site in this purified system is 25% of that observed in inverted membrane vesicles. Each functional site can catalyze multiple cycles of precursor translocation. These results indicate that the purified preprotein translocase properly reconstitutes translocation.

Adenosine Triphosphatases↗

PubMed1993

Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding.

Separation of unilamellar model membrane vesicles from external solution is often an important step in quantitation of vesicle bound or entrapped materials. An efficient method that allows pelleting of both small and large model membrane vesicles by centrifugation is described in this report. In this method streptavidin is added to vesicles containing a trace amount of biotinylated lipid. The resulting aggregation allows pelleting of the vesicles using an ordinary high-speed centrifuge. Control experiments show that the addition of streptavidin does not induce substantial vesicle fusion or leakage of substances trapped in the internal aqueous compartment of the vesicles. The method can accommodate different phospholipid compositions and lipid concentrations. Experiments with proteins that switch between hydrophilic and hydrophobic states show that the method can readily be used to monitor protein binding to vesicles.

Adenosine Triphosphatases↗

PubMed1992

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.

Adenosine Triphosphatases↗

PubMed2001

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein.

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.

Adenosine Triphosphatases↗