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W Wickner

Publications and source records attributed to W Wickner.

At least 37 records · Page 2Linked to original sources
PubMed1998

A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion.

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

Adenosine Triphosphatases↗
PubMed1998

The SecA subunit of Escherichia coli preprotein translocase is exposed to the periplasm.

SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.

Adenosine Triphosphatases↗
PubMed1997

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling.

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.

Adenosine Triphosphatases↗
PubMed1997

Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation.

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overexpressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the membrane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835-843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion. SecA also associates nonsaturably and unproductively with lipid. In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease-resistant and that remain so even upon subsequent membrane disruption. Unlike the [35S]SecA that inserts into the membrane at SecYEGDFyajC as it promotes preprotein translocation, lipid-associated [35S]SecA does not chase from its protease-resistant state upon the addition of excess SecA. The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the membrane during preprotein translocation, as well as the distinction between the membrane association of SecA at translocation sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement.

Adenosine Triphosphatases↗
PubMed1997

Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.

Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein. SecYE is the conserved functional 'core' of the SecYEG complex. Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation. The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC). Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme'. Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction. Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase.

Adenosine Triphosphatases↗
PubMed1997

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase.

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.

Adenosine Triphosphatases↗
PubMed1997

A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance.

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between salt-washed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as I(B)2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.

Adenosine Triphosphatases↗
PubMed1997

Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF).

Vacuole inheritance in yeast involves the formation of tubular and vesicular "segregation structures" which migrate into the bud and fuse there to establish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacuoles must be primed for docking by the action of Sec17p (alpha-SNAP) and Sec18p (NSF). Priming is necessary for both fusion partners. It produces a labile state which requires rapid docking in order to lead productively to fusion. In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p. Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.

Adenosine Triphosphatases↗
PubMed1996

Separable ATPase and membrane insertion domains of the SecA subunit of preprotein translocase.

The SecA subunit of preprotein translocase drives ATP-dependent translocation of preproteins across the bacterial inner membrane concomitant with cycles of membrane insertion and de-insertion (Economou, A., and Wickner, W. (1994) Cell 78, 835-843). We have identified the membrane-inserting region of SecA as a 30-kDa domain in the C-terminal third of the protein beginning at aminoacyl residue 610. Limited proteolysis in the absence of translocation ligands indicates that the SecA monomer is composed of two primary structural domains, the 30-kDa membrane-inserting domain and an N-terminal 65-kDa ATPase domain. This limited protease treatment of SecA results in constitutive ATPase activity, indicating that intramolecular constraints between the two domains may play a role in the regulation of ATP hydrolysis by SecA.

Adenosine Triphosphatases↗
PubMed1996

Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.

Acyl Coenzyme A↗
PubMed1996

Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles.

S. cerevisiae inherits its vacuole by projecting vacuole-derived membrane vesicles and tubules into the bud, where they fuse to establish the daughter vacuole. This homotypic fusion event can be assayed in vitro. It requires Sec17p and Sec18p, the homologs of the mammalian alpha-SNAP and NSF, which cooperate in multiple steps of membrane trafficking. We now report that Sec17p, Sec18p, and ATP are only needed for an early stage of the reaction that results in Sec17p release. Sec17p and Sec18p actions precede, and are needed for, the step employing the Ras-like GTPase Ypt7p. Sec18p-driven release of Sec17p can even precede vacuole docking, as it can occur prior to mixing of vacuoles and is insensitive to vacuole concentration. Sec17p and Sec18p thus may function in a predocking stage of the reaction, rather than in bilayer fusion per se.

Adenosine Triphosphatases↗
PubMed1996

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae.

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a "segregation structure") into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one "low molecular weight activity" (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.

Cell-Free System↗
PubMed1996

A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small cytosolic factor which inhibits vacuole inheritance.

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.

Adenosine Triphosphate↗
PubMed1995

SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF.

The SecA subunit of E. coli preprotein translocase promotes protein secretion during cycles of membrane insertion and deinsertion at SecYEG. This process is regulated both by nucleotide binding and hydrolysis and by the SecD and SecF proteins. In the presence of associated preprotein, the energy of ATP binding at nucleotide-binding domain 1 (NBD1) drives membrane insertion of a 30 kDa domain of SecA, while deinsertion of SecA requires the hydrolysis of this ATP. SecD and SecF stabilize the inserted state of SecA. ATP binding at NBD2, though needed for preprotein translocation, is not needed for SecA insertion or deinsertion.

Adenosine Triphosphatases↗
PubMed1995

The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance.

In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother-cell vacuole into the growing daughter cell during the S phase. These vesicles then fuse and form the daughter-cell organelle. We have described previously in vitro reactions of the formation of vacuole-derived segregation structures and of vacuole-vacuole fusion. Homotypic vacuole fusion requires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors. These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither. Here, we report that Ypt7p, a ras-like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is required on both partners during the in vitro vacuole fusion reaction. The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP-bound form of ras-like GTPases from membranes, or by antibodies specific for Ypt7p. The presence of anti-Ypt7p during the early stages of the reaction inhibits the development of cytosol- and ATP-independent intermediates. Although cytosol and ATP are no longer needed for the late stage of vacuole inheritance in vitro, the inhibition of this late stage by anti-Ypt7p or Gdi1p requires the continued presence of ATP and cytosol. Ypt7p is the first GTPase for which a direct role in organelle inheritance has been established.

Base Sequence↗