(A) Schematic of the four yeast vacuolar SNAREs, the soluble Q-SNAREs (sQx), and their deletion derivatives sQx3Δ lacking regions C-terminal to the +3 layer of the SNARE domain. (B) Structure of …
Source data file (PDB) for Figure 1C.
Purified full-length vacuolar SNAREs will spontaneously, though slowly, assemble into a parallel 4-helical coiled coil structure, which extends from the N-terminal (−7) layer of the SNARE domain to …
(A) Schematic of fluorescently labeled SNARE constructs used in this study. SNAREs were derivatized as described previously (Torng et al., 2020). Wild-type Qc contains a single Cys residue at 208 at …
Source data file (Excel) for Figure 2B.
(A) Schematic of fluorescently labeled SNARE constructs used in this study. (B–G) Supplementary data for panels (B-G) in Figure 2. Subfigures are presented with the same subfigure labels and …
gvSince proteoliposome fusion promoted by synthetic tethers is inhibited by all concentrations of Sec17, whereas HOPS-dependent fusion is only inhibited by very high Sec17 levels (Song and Wickner, …
(A) Fusion incubations, as described in 'Materials and methods', had Ypt7/R and Ypt7/QaQb proteoliposomes (1:8000 Ypt7:lipid molar ratio, 1:16,000 SNARE:lipid molar ratio), 2 μM Qc3Δ, and, where …
Source data file (Excel) for Figure 3A,B and C.
Fusion blocked by deletion of the C-terminal five layers of any Q-SNARE domain is restored by Sec17 and Sec18. (A) Ypt7/R- and Ypt7/QaQb, (B) Ypt7/R- and Ypt7/QaQc-tm, and (C) Ypt7/R- and …
(A–D) Fusion incubations, as described in 'Materials and methods', had Ypt7/R and Ypt7/Qa proteoliposomes (1:8000 Ypt7-TM:lipid molar ratio, 1:16,000 SNARE:lipid molar ratio), 50 nM HOPS, 2 μM sQb …
Source data file (Excel) for Figure 4A–L.
Fusion between Ypt7/R and (A–D) Ypt/Qa, (E–H) Ypt7/Qb, or (I–L) Ypt7/Qc-tm proteoliposomes. Fusion assays are described in Figure 4, repeated more than three times. Mean values and standard …
Ypt7/R and Ypt7/Qa proteoliposomes were co-incubated in the presence of HOPS and other fusion proteins or inhibitors, allowing resolution of an early, HOPS-dependent reaction stage from late, …
(A-H, J, K) Fusion between Ypt7/R and Ypt7/Qa proteoliposomes (1:8000 Ypt7-TM:lipid and 1:16,000 SNARE/lipid molar ratios) was assayed with 50 nM HOPS, 2 μM sQb3Δ, 2 μM Qc3Δ, the indicated …
Source data file (Excel) for Figure 5A–H,J and K.
We prepared proteoliposomes with Ypt7, with R or Qa, and with either no Sec17, with an equimolar (to SNAREs) TM-Sec17 that bears an N-terminal hydrophobic transmembrane anchor domain derived from …
Fusion incubations ('Materials and methods') had (A–C) Ypt7/R and Ypt7/Qa (w.t. (black), Gly mutant (blue), Ser mutant (red), or Ala mutant (green)) proteoliposomes (1:8000 Ypt7-TM:lipid molar …
Source data file (Excel) for Figure 6A–F.
(A–C) Fusion with 2 μM sQb and 2 μM Qc. (D–F) Fusion with 2 μM sQb3Δ and 2 μM Qc3Δ. Experiments were repeated in quadruplicate; mean values and standard deviations for fusion after 30 min are shown.
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 | GST-R (Nyv1) | PMID:18650938 | Purified from E. coli. | |
Peptide, recombinant protein | GST-Qa (Vam3) | PMID:18650938 | Purified from E. coli. | |
Peptide, recombinant protein | GST-Qb (Vti1) | PMID:18650938 | Purified from E. coli. | |
Peptide, recombinant protein | His-R (Nyv1) | PMID:22174414 | Purified from E. coli. | |
Peptide, recombinant protein | GST-sQa (soluble) | PMID:28637767 | Purified from E. coli. | |
Peptide, recombinant protein | MBP-sQb (soluble) | PMID:24088569 | Purified from E. coli. | |
Peptide, recombinant protein | GST-Qb (Vti1)3Δ | This study | Purified from E. coli. | |
Peptide, recombinant protein | GST-Qa (Vam3)3Δ | This study | Purified from E. coli. | |
Peptide, recombinant protein | Qc (Vam7) | PMID:17699614 | Purified from E. coli. | |
Peptide, recombinant protein | Qc (Vam7)-tm | PMID:23071309 | Purified from E. coli. | |
Peptide, recombinant protein | Qc (Vam7) C208S, M250C | This study | Purified from E. coli. | |
Peptide, recombinant protein | Qc (Vam7) C208S, A316C | This study | Purified from E. coli. | |
Peptide, recombinant protein | MBP-Cys-sQb (SNARE domain) | PMID:28637767 | Purified from E. coli. | |
Peptide, recombinant protein | MBP-Cys-sQb (SNARE domain) | PMID:28637767 | Purified from E. coli. | |
Peptide, recombinant protein | Ypt7 | PMID:24088569 | Purified from E. coli. | |
Peptide, recombinant protein | Ypt7-TM | PMID:31235584 | Purified from E. coli. | |
Peptide, recombinant protein | Sec17 | PMID:19414611 | Purified from E. coli. | |
Peptide, recombinant protein | Sec17 FSMS | PMID:28925353 | Purified from E. coli. | |
Peptide, recombinant protein | Sec17 KEKE | PMID:28925353 | Purified from E. coli. | |
Peptide, recombinant protein | Sec17 LALA | PMID:19414611 | Purified from E. coli. | |
Peptide, recombinant protein | GST-Sec17 6AtoN | This study | Purified from E. coli. | |
Peptide, recombinant protein | GST-Sec17-TM | PMID:28718762 | Purified from E. coli. | |
Peptide, recombinant protein | GST-Sec17-TM FSMS | PMID:28718762 | Purified from E. coli. | |
Peptide, recombinant protein | TEV protease | PMID:18007597 | Purified from E. coli. | |
Peptide, recombinant protein | HOPS | PMID:18385512 | Purified from Saccharomyces cerevisiae. | |
Antibody | Anti-Vam3 (rabbit polyclonal) | PMID:12566429 | Wickner lab stock | WB: 1:2000 |
Antibody | Anti-Nyv1 (rabbit polyclonal) | PMID:10385523 | Wickner lab stock | WB: 0.65 μg/ml |
Antibody | Anti-Vti1 (rabbit polyclonal) | PMID:18007597 | Wickner lab stock | WB: 0.47 μg/ml |
Antibody | Anti-Vam7 (rabbit polyclonal) | PMID:14734531 | Wickner lab stock | WB: 0.1 μg/ml |
Antibody | Anti-Vps16 (rabbit polyclonal) | PMID:18007597 | Wickner lab stock | WB: 1 μg/ml |
Antibody | Anti-Vps33 (rabbit polyclonal) | PMID:10944212 | Wickner lab stock | Inhibition assay: 1 μg |
Antibody | Anti-Sec18 (rabbit polyclonal) | PMID:11483507 | Wickner lab stock | Inhibition assay: 1 μg |
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 | |
Software and algorithms | UN-SCAN-IT | Silk Scientific | ||
Chemical compound, drug | Alexa Fluor 568 C5-maleimide | Thermo Fisher Scientific | A20341 | |
Chemical compound, drug | Oregon Green 488 Maleimide | Thermo Fisher Scientific | O6034 | |
Chemical compound, drug | Oregon Green 488 Maleimide | Thermo Fisher Scientific | O6034 | |
Chemical compound, drug | Pierce TCEP-HCl | Thermo Fisher Scientific | 20490 | |
Chemical compound, drug | L-cysteine | Sigma-Aldrich | 30089 |