Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, United States
Figures
Figure 1
Sec17, Sec18, and ATPγS promote rapid and homotypic fusion and vacuole protein sorting (HOPS)-independent fusion without SNARE zippering.
Proteoliposomes were prepared with vacuolar lipids, membrane-anchored Ypt7, and either R or the 3Q SNAREs with molar ratios of 1Ypt7:8000 lipids and 1 of each SNARE/16,000 lipids. Since Qc3Δ is a labile member of SNARE complexes (Song et al., 2021), a fivefold molar excess to the other SNAREs was used in preparing Ypt7/QaQbQc3Δ proteoliposomes. The initial mixtures at t = 0 were 18 μl, and remaining components were added at 30 min in 2 μl. Vertical dotted lines delineate the first 5 min, termed α, and the 2 min β interval after further additions.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Saccharomyces cerevisiae) | Nyv1 | Saccharomyces Genome Database | SGD:S000004083 | |
| Gene (Saccharomyces cerevisiae) | Vam3 | Saccharomyces Genome Database | SGD:S000005632 | |
| Gene (Saccharomyces cerevisiae) | Vti1 | Saccharomyces Genome Database | SGD:S000004810 | |
| Gene (Saccharomyces cerevisiae) | Vam7 | Saccharomyces Genome Database | SGD:S000003180 | |
| Gene (Saccharomyces cerevisiae) | Ypt7 | Saccharomyces Genome Database | SGD:S000004460 | |
| Gene (Saccharomyces cerevisiae) | Sec17 | Saccharomyces Genome Database | SGD:S000000146 | |
| Gene (Saccharomyces cerevisiae) | Sec18 | Saccharomyces Genome Database | SGD:S000000284 | |
| Gene (Saccharomyces cerevisiae) | Vps33 | Saccharomyces Genome Database | SGD:S000004388 | |
| Gene (Saccharomyces cerevisiae) | Vps39 | Saccharomyces Genome Database | SGD:S000002235 | |
| Gene (Saccharomyces cerevisiae) | Vps41 | Saccharomyces Genome Database | SGD:S000002487 | |
| Gene (Saccharomyces cerevisiae) | Vps16 | Saccharomyces Genome Database | SGD:S000005966 | |
| Gene (Saccharomyces cerevisiae) | Vps11 | Saccharomyces Genome Database | SGD:S000004844 | |
| Gene (Saccharomyces cerevisiae) | Vps18 | Saccharomyces Genome Database | SGD:S000004138 | |
| Peptide, recombinant protein | R (Nyv1) | PMID:21976702 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Qa (Vam3) | PMID:18650938 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Qb (Vti1) | PMID:21976702 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Qc (Vam7) | PMID:17699614 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Qc (Vam7) 3Δ | PMID:19414611 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Ypt7-TM | PMID:31235584 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Sec17 | PMID:19414611 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | Sec18 | PMID:8620540 | Purified from Escherichia coli. | |
| Peptide, recombinant protein | GST-PX | This Study | Purified from Escherichia coli. | |
| Peptide, recombinant protein | HOPS | PMID:18385512 | Purified from Saccharomyces cerevisiae | |
| Chemical compound,drug | Cy5-derivatized streptavidin | SeraCare Life Sciences | 5270–0023 | |
| Chemical compound,drug | Biotinylated PhycoE | Thermo Fisher Scientific | p811 | |
| Chemical compound,drug | Streptavidin | Thermo Fisher Scientific | 434302 | |
| Chemical compound,drug | 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine | Avanti polar lipids | 850385 | |
| Chemical compound,drug | 1,2-Dilinoleoyl-sn-glycero-3-phospho-L-serine | Avanti polar lipids | 840040 | |
| Chemical compound,drug | 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine | Avanti polar lipids | 850755 | |
| Chemical compound,drug | 1,2-Dilinoleoyl-sn-glycero-3-phosphate | Avanti polar lipids | 840885 | |
| Chemical compound,drug | L-α-Phosphatidylinositol | Avanti polar lipids | 840044 | |
| Chemical compound,drug | 1,2-Dipalmitoyl-sn-glycerol | Avanti polar lipids | 800816 | |
| Chemical compound,drug | Ergosterol | Sigma | 45480 | |
| Chemical compound,drug | PI(3)P | Echelon Bioscience | P-3016 | |
| Chemical compound,drug | Rhodamine DHPE | Invitrogen | L1392 | |
| Chemical compound,drug | NBD-PE | Invitrogen | N360 | |
| Chemical compound,drug | Marina-blue | Invitrogen | M12652 |
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Reagents were purchased, and proteins purified, as described in Song et al., 2021.
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Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS
eLife 10:e73240.
https://doi.org/10.7554/eLife.73240
