BILL WICKNER LAB at Dartmouth
PUBLICATIONS
2023
Orr, A., and Wickner, W. (2023) MARCKS effector domain, a reversible lipid ligand, illuminates late stages of membrane fusion. Mol Biol Cell DOI: 10.1091/mbc.E23-06-0228.
To study the fully assembled proteins which catalyze membrane fusion, we’ve long needed a reversible and late-stage fusion inhibitor. We’ve now found that the MARCKS Effector Domain peptide (MED) is just such a fusion inhibitor. It binds lipid to prevent the bilayer rearrangements of fusion, and the block caused by MED is easily reversed by either the MED ligand Ca:Calmodulin, by higher ionic strength to loosen the MED:lipid bond, or by extra energy for fusion from Sec17.
When SNARE complexes including the Qc-SNARE assemble during a blockade by MED (part A), they are no longer inhibited (after MED blockage reversal by calmodulin) by a large molar excess of a truncated Qc-SNARE which suffices to block if added along with Qc-SNARE from the start. Similarly, if the SNARE complex was formed with the truncated Qc during MED blockade, the addition of even a big molar excess of full-length functional Qc isn’t sufficient to restore fusion after reversal of the MED blockade (part B).
FIGURE 3: trans-SNARE assembly is stable during MED block.
Wickner, W., Lopes, K., Song, H., Rizo, J., and Orr, A. (2023) Efficient fusion requires a membrane anchor on the Qa SNARE. Mol. Biol. Cell DOI: 10.1091/mbc.E23-02-0052
Since the Qa and Qb SNARE are both anchored to the same membrane, and since they are adjacent in the 4-SNARE bundle, it seemed likely that they would have equivalent roles in fusion. However, we find that the Qa has to be anchored for fusion, and the Qb anchor is dispensible.
Anchored Qa-SNARE supports fusion but anchored Qb-SNARE does not (upper panel) unless the reactions also have Sec17 and Sec18. Assay of the R-SNARE which is bound to Qa-SNARE in trans (lower panel) shows that either anchored Qa- or Qb- SNARE can form trans-SNARE complex, and the amount of this complex isn’t affected by Sec17/Sec18.
Orr, A. and Wickner, W. (2023) PI3P regulates multiple stages of membrane fusion. Mol. Biol. Cell DOI: 10.1091/mbc.E22-10-0486
Phosphatidylinositol-3-Phosphate (PI3P) directly binds the Qc-SNARE and HOPS. Even when the Qc-SNARE had been pre-assembled into a complex with the other Q-SNAREs and HOPS was replaced by a synthetic tether, PI3P is still required for fusion.
With vacuolar mixed lipids (VML) which are 1% PI3P, even 10 nM Qc-SNARE supports full fusion (leftmost black bar). Fusion is lost if either the Qc SNARE has the Y42A mutation to block PI3P recognition (blue bar) or the PI3P is omitted from the VML mixture (red bar). With higher Qc concentrations, PI3P recognition isn’t required (right 3 bars).
Even when the three Q-SNAREs are pre-assembled into a complex on the membrane of one fusion partner, fusion with HOPS (panel A) or with a synthetic tether (panel B) requires PI3P (black symbols), suggesting PI3P functions beyond recruitment of HOPS or Qc.
2022
Orr, A., and Wickner, W. (2022) Fusion with wild-type SNARE domains is controlled by juxtamembrane domains, transmembrane anchors, and Sec17. Mol Biol Cell DOI: 10.1091/mbc.E21-11-0583.
Eight or nine amino acids, the juxtamembrane domains, connect the SNARE domains of the R- and Qa-SNAREs to their trans-membrane anchors. While fusion is fine when the transmembrane anchors are “swapped”, fusion is blocked if the juxtamembrane regions are swapped. This block is bypassed by Sec17 and Sec18. All this fits well with zippering extending beyond the SNARE domains and into the juxtamembrane domains, but that zippering isn’t needed for Sec17 to directly drive fusion.
Orr, A., and Wickner, W. (2022) Sec18 supports membrane fusion by promoting Sec17 membrane association. Mol Biol Cell DOI: 10.1091/mbc.e22-07-0274
Sec18 dramatically lowers the concentration of Sec17 needed for fusion when the SNAREs can’t normally zipper. Since Sec18 binds (via Sec17) to the N-terminal end of the SNARE domains, quite far from the site of fusion, how does it help out? Here we show that Sec18 binds several Sec17s, allowing the combined hydrophobicity of the N-terminal apolar loop of each Sec17 to drive the Sec18:3Sec17 complex to stably bind to lipids, delivering Sec17 to where it can interact with SNAREs.
2021
Song, H., Torng, T., Orr, A., Brunger, A.T., and Wickner, W. (2021) Sec17/Sec18 can support membrane fusion without help from completion of SNARE pairing. eLife, https://DOI.org/10.7554/eLife.67578.
While SNARE zippering alone can drive fusion, rapid fusion needs Sec17 and Sec18 too. Here, we present the striking finding that Sec17 and Sec18 can bind to the platform of partially-zippered SNAREs and drive fusion by themselves, even when each of the 3 Q—SNAREs have been modified so that there’s no possible energy to be gained from zippering!
Torng, T. and Wickner, W. (2021) Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion. Mol. Biol. Cell, doi: 10.1091/mbc.E21-07-0373.
Of the 8 vacuolar lipids, only two, phosphatidylinositol (PI) and phoslphatidylinositol-3-phosphate (PI3P), are essential to activate HOPS for catalysis of SNARE complex assembly. Strikingly, a minimal set of lipids (phosphatidylcholine, PI, PI3P, and ergosterol) support full HOPS-dependent assembly of trans-SNARE complexes (C and D below) but don’t support any fusion at all (B and D)! The other lipids are crucial for the bilayer rearrangements which constitute fusion of closely apposed membranes.
2020
Song, H., Orr, A., Lee, M., Harner, M., and Wickner, W. (2020) HOPS recognizes each SNARE, assembling ternary trans-SNARE complexes for rapid fusion upon engagement with the 4th SNARE. eLife DOI 10.7554/eLife.53559.
The key to understanding how HOPS catalyzes SNARE complex assembly is identifying intermediates on the assembly pathway. While even a synthetic tether (GST-PX) supports fusion if the 3 Q-SNAREs are preassembled (Panel I, A, B, red curves), HOPS is required if even one Q-SNARE isn't preassembled (Panel I, panels C, D, E, blue vs red curves). HOPS can form a rapid-fusion intermediate with the R-SNARE from one membrane, and either the Qa SNARE from the other or (Panel II) the Qb and Qc SNARE from the other. These "rapid-fusion" intermediates (Panel III), either HOPS:R:Qa (Panel III, intermediate 3) or HOPS:R:QbQc (intermediate 4), give sudden fusion when provided the missing SNARE(s) (Panel II, curve e).
Panel I
Panel II
Panel III
Torng, T., Song, H. and Wickner, W. (2020) Asymmetric Rab activation of vacuolar HOPS to catalyze SNARE complex assembly. Mol. Biol. Cell, in press. DOI: 10.1091/mbc.E20-01-0019.
A physical assay of assembled SNARE complex is presented, based on the FRET between a fluorphore bound to the Qb-SNARE and a different fluorophore bound to the Qc-SNARE. This assay allowed 3 striking findings: A. SNARE domain mutual affinities don't suffice for stable SNARE complex assembly; at least 2 of the SNAREs have to be co-localized to a common membrane or a common detergent micelle, shown here by the loss of FRET as high detergent dilutes the SNAREs among the micelles. B. HOPS is allosterically activated as an assembly catalyst for even soluble SNAREs when it is associated with both the Rab Ypt7 and with vacuolar lipids. C. HOPS is best stimulated as a SNARE-assembly catalyst by Ypt7 when the R-SNARE is on the same membrane as Ypt7.
Lee, M., Orr, A., Wickner, W., and Song, H. (2020) A Rab prenyl membrane-anchor allows effector recognition to be regulated by bound guanine nucleotide. Proc. Natl. Acad. Sci USA, DOI 10.1073/pnas.2000923117
Since most proteins use a long polypeptide segment to anchor to membranes, why do Rab GTPases use prenyl anchors? We show that the prenyl anchor affects the conformation of the vacuolar Rab Ypt7 so that its GTP- or GDP-bound state can be "read" by HOPS.
2019
Song, H. and Wickner, W. (2019) Tethering guides fusion-competent trans-SNARE assembly. Proc. Natl. Acad. Sci. USA, 116, 13952-13957; doi/10.1073/pnas.1907640116.
A synthetic tether of dimeric glutathione-S-transferase fused to a PX domain (which has direct affinity for PI3P) will tether two liposomes bearing PI3P. When the 3 Q-SNARE are preassembled on one fusion partner, and the R-SNARE is on the other, fusion needs tethering, and PI3P:PX-GST:GST-PX:PI3P works perfectly well.
Jun, Y., and Wickner, W. (2019) Sec17/aSNAP and Sec18/NSF restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments. Proc. Natl. Acad. Sci. USA doi 10.1073/pnas.1913985116.
There is some promiscuity to SM protein activation of SNAREs to form complexes for fusion, but the presence of Sec17, Sec18, and ATP will stimulate fusion when the R-SNARE, the Q-SNAREs, and the SM protein (or protein complex) are from the same compartment (ER or vacuole) but will block fusion if they are not.
2018
Harner, M. and Wickner, W. (2018) Assembly of intermediates for rapid membrane fusion. J. Biol. Chem. 293, 1346-1352.
A rapid fusion intermediate assembles with HOPS bridging membranes bearing Ypt7:R-SNARE and Ypt7:Qa,Qc-SNAREs. This is triggered to rapid fusion by the addition of Sec17 and the Qb-SNARE.
2017
Wickner, W. and Rizo, J. (2017) A cascade of multiple proteins and lipids catalyzes membrane fusion. Mol. Biol. Cell 28, 707-711, doi:10.1091/mbc.E16-07-0517.
A review of fusion, comparing vacuolar and neuronal synaptic fusion pathways.
Orr, A., Song, H., Rusin, S.F., Kettenbach, A.N., and Wickner, W. (2017) HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex. Mol. Biol. Cell 28, 975-983.
HOPS will allow liposomes with R- or Qa-SNARE and Ypt7 to assemble with the other 3 soluble-SNAREs to form stable 4-SNARE complex. A. Assay scheme, B. Assembly of each SNARE onto Ypt7,R- and Ypt7,Qa-proteoliposomes requires HOPS and each of the other SNAREs.
Song, H., Orr, A., Duan, M., Merz, A., and Wickner, W. (2017) Sec17/Sec18 act twice, enhancing fusion and then disassembling cis-SNARE complexes. eLife, https://doi.org/10.7554/eLife.26646.001.
Song, H. and Wickner, W. (2017) A short region upstream of the yeast vacuolar Qa SNARE heptad repeats promotes membrane fusion through enhanced SNARE complex assembly. Mol. Biol. Cell, 28, 2282-2289.
The 20 aminoacyl residues of the Qa N-domain which lie upstream of the SNARE domain are vital for fusion; the rest of the Qa N-domain, and all of the Qb N-domain, are dispensible.
2016
Zick, M. and Wickner, W. (2016) Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement. Mol. Biol. of the Cell 27, 2590-2597.
A. Fatty-acyl chains of physiological fluidity (18:2, 18:2) allow fusion at physiological levels of SNAREs (e.g. 1:32,000 molar ratio of SNAREs:lipid), while less fluid lipids (16:0, 18:1) need higher SNARE levels. B. Fusion with physiological SNARE levels requires GTP-activated Ypt7 (circles), while higher SNARE levels bypass this requirement (squares). Most fusion studies in the literature use very high SNARE levels which mask all the other requirements. C. With physiological SNARE levels (1:32,000) and fatty acyl chains (18:2, 18:2), Ypt7 is needed on both membranes, but bound GTP is only needed on the proteoliposomes bearing the R-SNARE.
2015
Orr, A., Wickner, W., Rusin, S.F., Kettenbach, A.N., and Zick, M. (2015) Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion. Mol. Biol. Cell 26, 305-315.
HOPS has affinity for acidic lipids and Ypt7, and can use either to support fusion. After phosphorylation to P-HOPS by the vacuolar kinase Yck3, it requires both acidic lipids and Ypt7 for fusion.
Zick, M., Orr, A., Schwartz, M.L., Merz, A.J., and Wickner, W.T. (2015) Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18. Proc. Natl. Acad. Sci. USA 112, E2290-97. doi:10.1073/pnas.1506409112.
When none of the SNAREs are initially in a complex (R and Qa SNAREs on proteoliposomes, Qb and Qc as soluble SNAREs), fusion requires Sec17.
Baker, R.W., Jeffrey, P.D., Zick, M., Phillips, B.P., Wickner, W.T., and Hughson, F.M. (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349, 1111-1114.
The Vps33 subunit of HOPS, an SM protein, has direct affinity for the R- and Qa- SNARE domains, binding them in parallel grooves on the protein.
2014
Zick, M., Stroupe, C., Orr, A., Douville, D., and Wickner, W. (2014) Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. eLife. doi: 10.7554/eLife.01879.
Omission of the non-bilayer prone lipids (PE, DAG, and Ergosterol) blocks fusion while allowing unimpeded trans-SNARE complex assembly.
Zick, M. and Wickner, W. (2014) A distinct tethering step is vital for vacuole membrane fusion. eLife, doi: 10.7754/eLife.03251.
This study drew 3 major conclusions: A. Lipid dequenching isn't a reliable assay for fusion. B. PI3P in trans from the PX domain of the Qc-SNARE can provide the tethering needed for HOPS-independent fusion of R- and 3Q-SNARE proteoliposomes. C. Even with substantial trans-SNARE pairing, HOPS is still needed for fusion.
2013
Karunakaran, V. and Wickner, W. (2013) Fusion proteins and select lipids cooperate as membrane receptors for the Soluble N-Ethylmaledimide-sensitive Factor Attachment Protein Receptor (SNARE) Vam7p. J. Biol. Chem 288, 28557-28566.
Vam7 binds membranes by its affinites for HOPS, PI3P, the other SNAREs, and even Sec17.
Zick, M. and Wickner, W. (2013) The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7p into fusogenic trans-SNARE complexes. Mol. Biol. Cell, 24, 3746-3753.
Vam7 at high concentrations supports fusion (A, black bars) unless deprived of its affinity for PI3P by the Y42A mutation (B). Added nonspecific tether PEG allows fusion at lower Vam7 levels (A, gray vs black bars), but HOPS allows fusion at even lower levels (open bars). Note that even with HOPS present, the lowest levels of Vam7 still rely on their PI3P affinity to support fusion (A vs B).
Wickner, W. (2013) Profile of Thomas Südhof, James Rothman, and Randy Schekman, 2013 Nobel Laureates in Physiology or Medicine. Proc. Natl. Acad. Sci. USA 46, 18349-18350.
A celebration of old friends, and of the field.
2012
Zick, M. and Wickner, W. (2012) Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion. Mol. Biol. Cell 23, 3429-3437.
Even with high SNARE levels and rigid fatty acyl chains, HOPS phosphorylation confers Rab nucleotide specificity.
Xu, H. and Wickner, W. (2012) N-terminal domain of the Vacuolar SNARE Vam7p promotes trans-SNARE complex assembly. Proc. Natl. Acad. Sci. USA 109, 17,936-17,941.
2011
Xu, H., Zick, M., Wickner, W., and Jun, Y. (2011) A lipid-anchored SNARE supports membrane fusion. Proc. Natl. Acad. Sci. USA 108, 17325-17330.
With a prenyl anchor replacing the trans-membrane anchor for Nyv1, fusion is good but requires HOPS, Sec17, and Sec18.
Wickner, W.T., Stubbe, J., Hirschberg, C.B., Garrett, T., and Dowhan, W. (2011) Chris Raetz, scientist and enduring friend. Proc. Natl. Acad. Sci. USA 108, 17255-17256.
Chris was my college and medical school roommate, and a dear friend.
Wickner, W. (2011). Eugene Patrick Kennedy, 1919-2011. Proc. Natl. Acad. Sci. USA 108, 19122-19123.
My teacher, and role model in science and in life.
Zucchi, P. and Zick, M. (2011) Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol. Biol. Cell 22, 4635-4646.
While showing that SNAREs and SNARE complexes enhance permeability to small molecules and even lead to lysis, we developed a rigorous assay for fusion and lysis:
2010
Hickey, C. and Wickner, W. (2010) HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly. Mol Biol. Cell 21, 2297-2305.
Quantitative study of the roles of HOPS, vacuolar lipids, and Ypt7 in tethering.
Xu, H., Jun, Y., Thompson, J., Yates, J., and Wickner, W. (2010) HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion. EMBO J. 29, 1948-1960.
A pioneering study with 2 major findings! Using intact vacuoles, it was shown that trans-associations led to Sec17:SNARE association, and in fact most trans-SNARE complex is bound up with Sec17! With proteoliposomes, it was shown that trans-SNARE complexes are only insensitive to Sec17/Sec18/ATP if the membranes are intact and HOPS is present (lanes 1-3 vs 4-6, below).
