Publications by authors named "William T Wickner"

18 Publications

  • Page 1 of 1

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.

Elife 2021 May 4;10. Epub 2021 May 4.

Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States.

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.
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http://dx.doi.org/10.7554/eLife.67578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143792PMC
May 2021

HOPS recognizes each SNARE, assembling ternary -complexes for rapid fusion upon engagement with the 4th SNARE.

Elife 2020 01 21;9. Epub 2020 Jan 21.

Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States.

Yeast vacuole fusion requires R-SNARE, Q-SNAREs, and HOPS. A HOPS SM-family subunit binds the R- and Qa-SNAREs. We now report that HOPS binds each of the four SNAREs. HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled, ushering them into a functional complex. Co-incubation of HOPS, proteoliposomes bearing R-SNARE, and proteoliposomes with any two Q-SNAREs yields a rapid-fusion complex with 3 SNAREs in a -assembly. The missing Q-SNARE then induces sudden fusion. HOPS can 'template' SNARE complex assembly through SM recognition of R- and Qa-SNAREs. Though the Qa-SNARE is essential for spontaneous SNARE assembly, HOPS also assembles a rapid-fusion complex between R- and QbQc-SNARE proteoliposomes in the absence of Qa-SNARE, awaiting Qa for fusion. HOPS-dependent fusion is saturable at low concentrations of each Q-SNARE, showing binding site functionality. HOPS thus tethers membranes and recognizes each SNARE, assembling R+Qa or R+QbQc rapid fusion intermediates.
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http://dx.doi.org/10.7554/eLife.53559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994237PMC
January 2020

A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly.

Science 2015 Sep;349(6252):1111-4

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast homotypic fusion and vacuole protein-sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.
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http://dx.doi.org/10.1126/science.aac7906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727825PMC
September 2015

Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18.

Proc Natl Acad Sci U S A 2015 May 20;112(18):E2290-7. Epub 2015 Apr 20.

Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755; and

Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17(L291A,L292A) did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17(F21S,M22S), with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17(F21S,M22S), interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.
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http://dx.doi.org/10.1073/pnas.1506409112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426435PMC
May 2015

A distinct tethering step is vital for vacuole membrane fusion.

Elife 2014 Sep 25;3:e03251. Epub 2014 Sep 25.

Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, United States.

Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.
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http://dx.doi.org/10.7554/eLife.03251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200421PMC
September 2014

Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

Elife 2014 Jan 1;3:e01879. Epub 2014 Jan 1.

Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, United States.

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937803PMC
http://dx.doi.org/10.7554/eLife.01879DOI Listing
January 2014

Profile of Thomas Sudhof, James Rothman, And Randy Schekman, 2013 Nobel Laureates in Physiology or Medicine.

Proc Natl Acad Sci U S A 2013 Nov 24;110(46):18349-50. Epub 2013 Oct 24.

Biochemistry Department, Dartmouth Medical School, Dartmouth College, Hanover, NH 03755.

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http://dx.doi.org/10.1073/pnas.1319309110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832004PMC
November 2013

Christian Raetz: scientist and friend extraordinaire.

Annu Rev Biochem 2013 7;82:1-24. Epub 2013 Mar 7.

Department of Biochemistry and Molecular Biology and Center for Membrane Biology, University of Texas Medical School, Houston, Texas 77030, USA.

Chris Raetz passed away on August 16, 2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gram-negative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpinnings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiosity-driven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues.
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http://dx.doi.org/10.1146/annurev-biochem-012512-091530DOI Listing
November 2013

N-terminal domain of vacuolar SNARE Vam7p promotes trans-SNARE complex assembly.

Proc Natl Acad Sci U S A 2012 Oct 15;109(44):17936-41. Epub 2012 Oct 15.

Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.

SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R- and Q-SNAREs. This function of the Vam7p N-domain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain.
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http://dx.doi.org/10.1073/pnas.1216201109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497749PMC
October 2012

Eugene Patrick Kennedy, 1919-2011.

Proc Natl Acad Sci U S A 2011 Nov 18;108(48):19122-3. Epub 2011 Nov 18.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

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http://dx.doi.org/10.1073/pnas.1117398108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228477PMC
November 2011

A lipid-anchored SNARE supports membrane fusion.

Proc Natl Acad Sci U S A 2011 Oct 10;108(42):17325-30. Epub 2011 Oct 10.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.
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http://dx.doi.org/10.1073/pnas.1113888108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198343PMC
October 2011

Chris Raetz, scientist and enduring friend.

Proc Natl Acad Sci U S A 2011 Oct 3;108(42):17255-6. Epub 2011 Oct 3.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

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http://dx.doi.org/10.1073/pnas.1114405108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198341PMC
October 2011

Trans-SNARE complex assembly and yeast vacuole membrane fusion.

Proc Natl Acad Sci U S A 2007 May 14;104(21):8755-60. Epub 2007 May 14.

Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.

cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p (alpha-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyv1p or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)-effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or elevated concentrations of Sec17p, which can displace HOPS from SNARE complexes, permit full trans-SNARE pairing but block fusion. These findings suggest that efficient fusion requires trans-SNARE complex associations with factors such as HOPS and subsequent regulated lipid rearrangements.
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http://dx.doi.org/10.1073/pnas.0702290104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885575PMC
May 2007

Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast.

J Biol Chem 2006 May 24;281(20):14523-8. Epub 2006 Mar 24.

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844, USA.

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.
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http://dx.doi.org/10.1074/jbc.M600911200DOI Listing
May 2006

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion.

EMBO J 2005 May 5;24(10):1775-86. Epub 2005 May 5.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.
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http://dx.doi.org/10.1038/sj.emboj.7600658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142591PMC
May 2005

Resolution of organelle docking and fusion kinetics in a cell-free assay.

Proc Natl Acad Sci U S A 2004 Aug 30;101(32):11548-53. Epub 2004 Jul 30.

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

In vitro assays of compartment mixing have been key tools in the biochemical dissection of organelle docking and fusion. Many such assays measure compartment mixing through the enzymatic modification of reporter proteins. Homotypic fusion of yeast vacuoles is measured with a coupled assay of proteolytic maturation of pro-alkaline phosphatase (pro-ALP). A kinetic lag is observed between the end of docking, marked by the acquisition of resistance to anti-SNARE reagents, and ALP maturation. We therefore asked whether the time taken for pro-ALP maturation adds a kinetic lag to the measured fusion signal. Prb1p promotes ALP maturation; overproduction of Prb1p accelerates ALP activation in detergent lysates but does not alter the measured kinetics of docking or fusion. Thus, the lag between docking and ALP activation reflects a lag between docking and fusion. Many vacuoles in the population undergo multiple rounds of fusion; methods are presented for distinguishing the first round of fusion from ongoing rounds of fusion. A simple kinetic model distinguishes between two rates, the rate of fusion and the rate at which fusion competence is lost, and allows estimation of the number of rounds of fusion completed.
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http://dx.doi.org/10.1073/pnas.0404583101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC511018PMC
August 2004

Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen.

J Cell Biol 2004 Jan;164(2):195-206

Dept. of Biochemistry, 7200 Vail Bldg., Dartmouth Medical School, Hanover, NH 03755-3844, USA.

Ca2+ transients trigger many SNARE-dependent membrane fusion events. The homotypic fusion of yeast vacuoles occurs after a release of lumenal Ca2+. Here, we show that trans-SNARE interactions promote the release of Ca2+ from the vacuole lumen. Ypt7p-GTP, the Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of which function during docking, are required for Ca2+ release. Inhibitors of SNARE function prevent Ca2+ release. Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2+ release. Vacuoles lacking either of two complementary SNAREs, Vam3p or Nyv1p, fail to release Ca2+ upon tethering. Mixing these two vacuole populations together allows Vam3p and Nyv1p to interact in trans and rescues Ca2+ release. Sec17/18p promote sustained Ca2+ release by recycling SNAREs (and perhaps other limiting factors), but are not required at the release step itself. We conclude that trans-SNARE assembly events during docking promote Ca2+ release from the vacuole lumen.
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http://dx.doi.org/10.1083/jcb.200310105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172329PMC
January 2004