Research | vacuole fusion

CURRENT RESEARCH

Membrane fusion is essential for neurotransmission, hormone secretion, and cell growth. Its mechanisms are utterly conserved throughout biology, among organelles at each stage of exocytic and endocytic vesicular traffic and among organisms from yeast to flies and worms to plants to humans. In light of this conservation, we choose to study fusion in the system with the very best cytology, genetics, and biochemistry, the homotypic fusion (to each other) of the vacuoles of baker's yeast (1-14). Here's a movie of a single yeast cell with a few clustered vacuoles which are visualized in 3 channels: by a green fluorescent protein-tagged vacuole membrane protein (left channel), by a vacuole vital dye (middle channel), and by Nomarski optics (right channel). Even a pretty low-tech light microscope allowed us to watch the fusion in vivo!

GFP-tagged vacuole protein

Vacuolar dye

Nomarski

Figure 1. Watch 4 vacuoles in 1 cell fuse, visualized in 3 fluorescence and light microscopy channels!

Glossary

Docking: Vacuole tethering, assembly of microdomains, & trans-SNARE associations.

Fusion: Mixing of lumens & membrane constituents without lysis.

SNARE: Proteins with conserved heptad repeat domains which associate in cis (all anchored to one bilayer) or trans (on apposed bilayers) in 4-helical coiled-coils

Qa: Vacuolar Vam3 SNARE

Qb: Vacuolar Vti1 SNARE

Qc: Vacuolar Qc SNARE, it has a PX domain with affinity for PI(3)P as a membrane anchor.

R: Vacuolar Nyv1 SNARE

Ypt7: The vacuolar Rab-family GTPase

HOPS: A complex of Vps11, 16, 18, 33, 39, and 41p; Rab effector, PIPx affinity, SNARE-assembly catalyst

Sec17: Yeast a-SNAP

Sec18: Yeast NSF

Tethering: 1st association between membranes

To complement the excellent genetics of vacuole fusion (15) and easy vacuole visualization, we've really developed vacuole fusion biochemistry, with isolated organelles (16) and with purified proteins. We make assays that are easy to do, that turn color or fluoresce, and are quantitative. Come to the lab in the morning, mix components, have coffee while fusion occurs and machines record the signal, and have Data By Lunch! That's the life. With this, we've defined the many proteins and lipids required for fusion (17-20), then reconstituted fusion with proteoliposomes bearing all-purified proteins (4, 21, 22), allowing us to address molecular mechanisms. This is kind of amazing!! No other fusion system offers all these tools; for example, other fusing membrane vesicles are often too small to see in light microscopes and watch in real-time, and other membranes are hard to isolate in a functional form for fusion (e.g., synaptic vesicles); for all these reasons, no other membrane fusion system has been studied at this depth! This was the work of our lab over the last 30 years (see Alumni section) and now, with all this done, we're in the era of Actually Figuring Out How It Works! Intense excitement!!

Fusion in every realm of biology, from yeast vacuoles to human neuronal synapses, requires prenyl-anchored Rab-family GTPases, their effector tethering complexes, SNAREs, and SNARE chaperones such as the Sec1/Munc 18 (SM) proteins, Sec17/aSNAP, and Sec18/NSF. Heptad-repeat SNARE domains assemble into 4-helical coiled-coils, with inward-facing apolar residues except for a central R or Q residue. SNAREs are in 4 families, termed R, Qa, Qb, and Qc, and assemble RQaQbQc complexes, in cis if anchored to one membrane or in trans if anchored to apposed membranes. Here are these components for yeast vacuoles:

Lipids:

Ergosterol stablizes microdomains

Diacylglycerol, ergosterol, and PE promote nonbilayer lipid phases for fusion.

PI(3)P helps SNARE localization via the Vam7p PX domain

PA promotes binding of HOPS and Sec18p

Proteins:

SNAREs can “snare” each other, in cis or in trans, destabilize bilayers, enrich fusion-prone lipids, and serve as a binding platform

Sec18p and Sec17p are an ATP-driven chaperone system that disassembles SNARE complexes.

Sec17 binds trans-SNAREs to trigger fusion.

Ypt7 is a Rab GTPase.

It works through…

HOPS, a heterohexameric complex which does it all!

a. Binds vacuoles through direct affinity for Ypt7p:GTP.

b. Direct affinity for phosphoinositides and acidic lipids

c. Direct affinity for each of the SNAREs

d. Its SM-family subunit binds the R- and Q-SNAREs, in parallel and in register

e. It catalyzes SNARE complex assembly

f. A member of the trans-SNARE complex, with direct affinity for Sec17

Figure 2. The lipids and proteins needed for vacuole fusion, whether in the intact cell, with the purified organelle, or as reconstituted in the proteoliposome with all the purified components!

Yeast vacuole fusion occurs in distinct stages: 1. Vacuolar cis-SNARE complexes are disassembled by Sec18/Sec17/ATP (23-25). 2. Vacuoles tether by association of the hexameric HOPS complex with the Rab Ypt7 on each partner membrane (22, 26). 3. SNAREs, Ypt7, HOPS, and key lipids become enriched in a microdomain surrounding the apposed surfaces of tethered membranes (27, 28). 4. HOPS is allosterically activated by Ypt7 and vacuolar lipids to catalyze SNARE assembly (13), first binding to the R-SNARE in cis to the Ypt7 receptor of HOPS, then to the Q-SNAREs in trans (13). During catalyzed SNARE assembly, the R- and Qa SNARE domains whose apolar surfaces initially face into grooves on the Vps33 SM-family subunit of HOPS (3) are released to associate with the other SNAREs in the assembling coiled coils SNARE bundle. 5. SNARE zippering and the membrane insertion of the Sec17 apolar loop (7) combine with the enrichment of nonbilayer-prone lipids (29) to lower the energy barrier for the lipid rearrangements of fusion (2, 7). Figure 3 depicts our working model of the pre-fusion complex.

Figure 3. Working model of pre-fusion complex

Our lab developed this system in its entirety, from 1986-present! Our findings are directly relevant to human disease; human HOPS is central to ARC syndrome (30) and is required for infection by Marburg and Ebola viruses (31) and Coxiella burnetii (32).

In the same year when it was shown that high levels of SNAREs can drive fusion (33), we detergent- solubilized vacuoles, fractionated the extracted proteins, and reconstituted fractions into proteoliposomes for assays of fusion (34). We found that Ypt7 is also essential for fusion when SNAREs are at physiological levels (34). We have pursued our reductionist path, purifying each required protein and reconstituting proteoliposomal fusion with vacuolar lipids (35) and with proteins at physiological concentrations to recapitulate (4) the known requirements for fusion in vivo. Our analysis has made substantial contributions to the evolution of the membrane fusion paradigm (Box 1).

“Classical” Model of Membrane Fusion

1. Fusion is driven by SNAREs alone.

2. Lipid bilayers are passive substrates

during fusion.

3. Tethering just concentrates SNAREs,

allowing them to assemble in trans.

4. SNAREs assemble spontaneously,

with SM recognition of Qa-SNAREs.

5. Sec17 (aSNAP) & Sec18 (NSF) are

only needed between fusion rounds

to disassemble SNAREs.

6. SNAREs confer organelle specificity.

7. Rabs simply localize effectors for tethering.

Lessons from Our Reconstituted Vacuole Fusion

1. Fusion requires Ypt7:GTP, HOPS, 4 SNAREs, Sec17, Sec18.

2. PIPx and acidic lipids bind peripheral proteins; fatty acyl

fluidity and non-bilayer prone lipids allow lipid rearrangements

3. Tethering allows trans-SNARE assembly in the right conformation for fusion.

4. HOPS binds each SNARE & catalyzes SNARE complex assembly

5. Sec17 and Sec18 act twice, to stimulate fusion per se and to

disassemble cis-SNARE complexes after fusion.

6. In the presence of Sec17/aSNAP and Sec18/NSF, ER vs vacuole

organelle specificity requires a tripartite match of R-SNAREs, 3Q-SNAREs, and SM protein or SM complex.

7. Rab & key lipids allosterically activate HOPS, asymmetrically

engaging R-SNAREs and catalyzing 4-SNARE assembly.

Box 1. Revising the membrane fusion paradigm

Here is our current working model of fusion. HOPS first mediates membrane tethering (Step1) by its affinities for the Rab GTPase Ypt7 on each membrane. It’s activated by binding to vacuole lipids and to Ypt7 to catalyze the first stage of zippering of the 4 SNAREs (Step2) to form a partially-zippered complex. This can then undergo slow fusion, perhaps limited by the slow dissociation of HOPS to “get out of the way” or by slow completion of zippering against the spring-like force of the membranes as they’re pulled together. Sec17 and Sec18 strongly stimulate the rate of fusion, first by Sec17 displacing HOPS (Step3), then joined by Sec18 (Step4) to form the fully assembled pre-fusion complex. This will promote zippering, but in addition Sec17 will promote fusion even without zippering. Normally, they work together. After fusion, ATP hydrolysis by Sec18 disassembles the structure to allow a further round.

OK, if you've read this far, you've deduced that this isn't just a "job"-- this is our passion, it's a calling, it's been the creation of labmates over the last 30 years, who've gone on to stellar careers of their own (see Alumni section).

Two Exciting Advances in the last few months!

2. Direct real-time assay of trans-SNARE association. The FRET between fluorescent Qb and fluorescent Qc is supported by Ypt7/R and Ypt7/Qa proteoliposomes in the presence of HOPS (red squares). Equivalent FRET is seen when fluorescent Qc lacks its 3 C-terminal heptads (green squares), which entirely blocks fusion (Fig1A, blue). These are therefore trans-SNARE complexes. [Cartoon; Ypt7/R: Ypt7:Qa proteoliposomes]. All samples include sQb and either Qc or QcD3, labeled with fluorophores which can give FRET. This ability to assay trans-SNARE complexes in real-time is a technical tool long sought in our lab!

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1. 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. PMCID: PMC4294677

2. 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. PMCID: PMC4426435

3. 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. PMCID: PMC4727825

4. 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. PMCID: PMC4985260

5. 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. PMCID: PMC5349777

6. 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. PMCID: PMC5385495

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11. 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. PMCID: PMC8876204

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