The nascent-polypeptide-associated complex: having a "NAC" for fidelity in translocation.
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Biological Transport↗
Biomedical subjects
W Wickner
Publications and source records attributed to W Wickner.
SecYEG and SecA are the stoichiometric components of preprotein translocase.
The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.
Adenosine Triphosphatases↗
On the road to translocase.
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Adenosine Triphosphatases↗
SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion.
SecA, the peripheral subunit of E. coli preprotein translocase, alternates between a membrane inserted and a deinserted state as part of the catalytic cycle of preprotein translocation. When SecA is complexed with SecY/E and preprotein, ATP drives a profound conformational change, leading to membrane insertion of a 30 kDa domain of SecA. The inserted domain is protease-inaccessible from the cytosolic side of the membrane, but becomes accessible upon membrane disruption. Concomitant with 30 kDa domain insertion, approximately 20 aminoacyl residues of the preprotein are translocated. Additional ATP, which may be hydrolyzed at the second ATP site of SecA, releases the translocated preprotein and allows the 30 kDa domain to deinsert, whence it can exchange with cytosolic SecA. Thus, SecA is the mobile subunit of an integral membrane transporter, consuming ATP during both the insertion and deinsertion phases of its catalytic cycle while guiding preprotein segments across the membrane.
Adenosine Triphosphatases↗
Band 1 subunit of Escherichia coli preportein translocase and integral membrane export factor P12 are the same protein.
Escherichia coli preprotein translocase consists of the peripheral membrane protein SecA and the integral membrane domain SecY/E. SecY/E, whether isolated chromatographically or by immunoprecipitation, was found to be complex of three polypeptides, SecY, SecE, and band 1. Band 1 did not correspond to a known sec gene product. The independent purification of the separate integral membrane polypeptides needed for reconstitution of translocation yielded SecY, SecE, and a protein that we termed P12. Based on the sequence of P12, we have prepared antisera to a carboxyl-terminal peptide domain and shown that this antiserum specifically labels only P12 on immunoblots of inner membrane vesicles. We now report that affinity-purified anti-P12 antibodies specifically label the band 1 subunit of the SecY/E complex, whether the SecY/E was isolated chromatographically or by precipitation with antibodies to an epitope-tagged SecY subunit. In addition, the antiserum to P12 can specifically immunoprecipitate the full three-subunit SecY/E complex from detergent extracts. This finding completes the identification of the polypeptides that are essential for catalysis of preprotein translocation.
Adenosine Triphosphatases↗
SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.
Mutations in secD and secF show impaired protein translocation across the inner membrane of Escherichia coli. We investigated the effect of SecD and SecF (SecD/F) depletion on preprotein translocation into inverted inner membrane vesicles (IMVs). Both IMVs and cells which were depleted of SecD/F were defective in their ability to maintain a proton electrochemical gradient. The translocation of pre-maltose binding protein (preMBP), which is strongly delta microH+ dependent, showed a 5-fold decreased rate with IMVs lacking SecD/F. In contrast, proteolytic processing of preMBP to MBP by leader peptidase was similar in IMVs containing and lacking SecD/F, consistent with earlier findings that only ATP-dependent translocation is required for the initiation of translocation. In the absence of a delta microH+, with ATP as the sole energy source, preMBP translocation into IMVs which contained or were depleted of SecD/F was identical. Translocation of the precursor of outer membrane protein A (proOmpA) in the presence of subsaturating ATP also required a generated delta microH+ and, under these conditions, proOmpA translocation required SecD/F. With saturating concentrations of ATP, where delta microH+ has little effect on in vitro proOmpA translocation, SecD/F also had little effect on translocation. These results explain why SecD/F effects are precursor protein dependent in vitro.
ATP-Binding Cassette Transporters↗
G-protein ligands inhibit in vitro reactions of vacuole inheritance.
During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.
Amino Acid Sequence↗
Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro.
Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted in vitro using isolated organelles, cytosol, and ATP. Using the requirements of the reaction and its susceptibility to inhibitors, we have divided the in vitro reaction into four biochemically distinct, sequential subreactions. Stage I requires exposure of vacuoles to solutions of moderate ionic strength. Stage II requires "stage I" vacuoles and cytosol. In stage III, stage II vacuoles react with ATP. Finally, during stage IV, stage III vacuoles at a certain, minimal concentration complete the fusion reaction without further requirement for any soluble components. Reagents that inhibit the overall vacuole inheritance reaction block distinct stages. Stage III of the reaction is sensitive to the proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A1, and to the ATP-hydrolyzing enzyme apyrase, suggesting that an electrochemical potential across the vacuolar membrane is required during this stage. Inhibition studies with the amphiphilic peptide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins might also be involved during this stage. Microcystin-LR, a specific inhibitor of protein phosphatases of type 1 and 2A, inhibits stage IV of the inheritance reaction, indicating that a protein dephosphorylation event is necessary for fusion. The definition of these four stages may allow the development of specific assays for the factors which catalyze each of the consecutive steps of the in vitro reaction.
Adenosine Triphosphate↗
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↗
Translocation can drive the unfolding of a preprotein domain.
Precursor proteins are believed to have secondary and tertiary structure prior to translocation across the Escherichia coli plasma membrane. We now find that preprotein unfolding during translocation can be driven by the translocation event itself. At certain stages, translocation and unfolding can occur without exogenous energy input. To examine this unfolding reaction, we have prepared proOmpA-Dhfr, a fusion protein of the well studied cytosolic enzyme dihydrofolate reductase (Dhfr) connected to the C-terminus of proOmpA, the precursor form of outer membrane protein A. At an intermediate stage of its in vitro translocation, the N-terminal proOmpA domain has crossed the membrane while the folded Dhfr portion, stabilized by its ligands NADPH and methotrexate, has not. When the ligands are removed from this intermediate, translocation occurs by a two-step process. First, 20-30 amino acid residues of the fusion protein translocate concomitant with unfolding of the Dhfr domain. This reaction requires neither ATP, delta mu H+ nor the SecA subunit of translocase. Strikingly, this translocation accelerates the net unfolding of the Dhfr domain. In a second step, SecA and ATP hydrolysis drive the rapid completion of translocation. Thus energy derived from translocation can drive the unfolding of a substantial protein domain.
Adenosine Triphosphatases↗
The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids.
To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr). A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein. Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain. After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase. Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking. The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit.
Adenosine Triphosphatases↗
The role of the mature domain of proOmpA in the translocation ATPase reaction.
The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel, E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either 53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus, the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase.
Adenosine Triphosphatases↗
Preprotein translocation creates a halide anion permeability in the Escherichia coli plasma membrane.
The electrochemical potential drives the translocation of the precursor form of outer membrane protein A (proOmpA) and other proteins across the plasma membrane of Escherichia coli. We have measured the electrical potential, delta psi, across inverted membrane vesicles during proOmpA translocation. delta psi, generated by the electron transport chain, is substantially dissipated by proOmpA translocation. delta psi dissipation requires SecA, ATP, and proOmpA. proOmpA which, due to the covalent addition of a folded protein to a cysteinyl side chain, is arrested during its translocation, can nevertheless cause the loss of delta psi. Thus the movement of charged amino acyl residues is not dissipating the potential. This translocation-specific reduction in delta psi is only seen in the presence of halide anions, although halide anions are not needed for proOmpA translocation per se. We therefore propose that translocation intermediates directly increase the membrane permeability to halide anions.
Anions↗
SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase.
The preprotein translocase of Escherichia coli is a multisubunit enzyme with two domains, the peripheral membrane protein SecA and the membrane-embedded SecY/E protein. SecY/E has been isolated as a complex of three polypeptides, SecY, SecE, and band 1. We now present four lines of evidence that the active species of SecY/E is composed of a tightly associated complex of these three subunits: 1) antibodies to SecY efficiently precipitate SecY/E activity as well as all three polypeptides; 2) the proportions of SecY, SecE, and band 1 in the immunoprecipitates are the same as in the starting fraction; 3) the immunoprecipitable complex is not disrupted by treatment with either high salt or urea but is disrupted by brief incubation at 20 degrees C, and the kinetics of dissociation of both band 1 and SecE from SecY at 20 degrees C parallel the loss of translocation ATPase activity; 4) upon immunoprecipitation of similar units of activity of translocase from detergent solutions from either wild-type membranes or a SecY and SecE overproducer strain, the SecE and band 1 subunits are recovered in the same proportions. These data establish that the subunits of SecY/E are firmly associated and that it is the associated complex which is active for translocation.
Adenosine Triphosphatases↗
Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting.
Cell division requires an accurate partitioning of cytoplasmic organelles. The segregation of vacuoles in the budding yeast Saccharomyces cerevisaie occurs at a specific time in the cell cycle and is spatially targeted to the small bud. Several yeast vac mutants have been isolated which are defective in this process. We have now cloned the VAC1 gene, corresponding to the first of these mutants, vac1-1. This gene encodes a protein of 515 amino acids, without homolog in the current data bases. It contains neither long hydrophobic stretches nor a classical leader peptide. The most notable aspect of the sequence is the presence of three zinc fingers. Yeast in which the VAC1 gene has been entirely deleted are viable. However, they grow more slowly than wild-type cells and only form microcolonies when grown on glycerol at 37 degrees C. These yeast are defective in vacuole segregation at both the permissive and nonpermissive temperatures. The vac1 mutant was previously shown to mislocalize carboxypeptidase Y to the cell surface, suggesting that Vac1p is involved in more than one vesicular traffic pathway.
Adaptor Proteins, Signal Transducing↗
In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae.
Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.
Adenosine Triphosphate↗
SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.
Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.
Adenosine Triphosphatases↗
Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase.
The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation. A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed. This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps.
Adenosine Triphosphatases↗