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W Wickner

Publications and source records attributed to W Wickner.

At least 19 recordsLinked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.

Adenosine Triphosphatases↗
PubMed1990

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

Bacterial Outer Membrane Proteins↗
PubMed1990

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation.

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.

Adenosine Triphosphatases↗
PubMed1990

The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.

The ATPase activity of SecA is stimulated by E. coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA. This activity is termed "translocation ATPase". Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes. We define the stimulation of SecA ATPase by lipid as "SecA/lipid ATPase". SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism. ProOmpA and ATP stabilize liposome-bound SecA. Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice. Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains.

Adenosine Triphosphatases↗
PubMed1990

ProOmpA contains secondary and tertiary structure prior to translocation and is shielded from aggregation by association with SecB protein.

Escherichia coli protein export involves cytosolic components termed molecular chaperones which function to stabilize precursors for membrane translocation. It has been suggested that chaperones maintain precursor proteins in a loosely folded state. We now demonstrate that purified proOmpA in its translocation component conformation contains both secondary and tertiary structure as analyzed by circular dichroism and intrinsic tryptophan fluorescence. Association with one molecular chaperone, SecB, subtly modulates the conformation of proOmpA and stabilizes it by inhibiting aggregation, permitting its translocation across inverted E.coli inner membrane vesicles. These results suggest that translocation competence does not simply result from the maintenance of an unfolded state and that molecular chaperones can stabilize precursor proteins by inhibiting their oligomerization.

Bacterial Outer Membrane Proteins↗
PubMed1990

Solubilization and functional reconstitution of the protein-translocation enzymes of Escherichia coli.

The SecY protein and other membrane proteins of Escherichia coli were solubilized by mixed micelles of n-octyl beta-D-glucopyranoside, phospholipids, and glycerol. Proteoliposomes formed from this extract by detergent dialysis supported energy-dependent translocation and processing of pro-OmpA. Translocation required ATP, SecY, and SecA and was stimulated by a proton-motive force. These results provide an important assay for the isolation and identification of membrane components involved in protein translocation.

Bacterial Outer Membrane Proteins↗
PubMed1990

Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.

Amino Acid Isomerases↗
PubMed1989

Export of prepro-alpha-factor from Escherichia coli.

Yeast prepro-alpha-factor translocates posttranslationally into yeast microsomes in vitro. This process is strongly influenced by the extreme carboxyl-terminal region of the protein. These features contrast with the properties of most eucaryotic proteins which are translocated into the endoplasmic reticulum. We have extended these studies by introducing the gene for the wild-type and several mutant forms of prepro-alpha-factor into Escherichia coli. Prepro-alpha-factor is secreted into the periplasm and processed to pro-alpha-factor. Its translocation across the plasma membrane requires the membrane potential and the secY gene product. Deletion mutant analysis showed that features of the pro-segment were essential for secretion of prepro-alpha-factor in E. coli, while the carboxyl-terminal region, which is required in yeast, is dispensible in E. coli. Neither size nor the presence of a unique topogenic sequence was sufficient to explain the requirement for the pro-segment.

Chromosome Deletion↗
PubMed1989

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

Amino Acid Isomerases↗