Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering
- Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, United States
- Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology Stanford University, United States
Figures
Figure 1 with 1 supplement
Model of the Sec18/Sec17/vacuolar SNARE complex and Sec17 mutations.
(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 …
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Figure 1—source data 1
Source data file (PDB) for Figure 1C.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig1-data1-v2.zip
Figure 1—figure supplement 1
SNARE associations during partial zippering.
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 …
Figure 2 with 2 supplements
Sec17 modifies SNARE conformation in a HOPS- and zippering-dependent manner and stabilizes complexes with truncated SNARE domains.
(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 …
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Figure 2—source data 1
Source data file (Excel) for Figure 2B.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Statistics and representative kinetic data for Figure 2.
(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 …
Figure 2—figure supplement 2
Direct binding of Sec17 to HOPS.
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, …
Figure 3 with 1 supplement
Fusion is blocked by deletion of the last five C-terminal SNARE domain layers from any single Q-SNARE and is restored by Sec17, Sec18, and ATP or ATPγS.
(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 …
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Figure 3—source data 1
Source data file (Excel) for Figure 3A,B and C.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Statistics for Figure 3.
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 …
Figure 4 with 2 supplements
Fusion with single membrane-anchored Q-SNAREs.
(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 …
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Figure 4—source data 1
Source data file (Excel) for Figure 4A–L.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Statistics data for Figure 4.
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 …
Figure 4—figure supplement 2
Selective inhibitors reveal successive fusion intermediates.
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, …
Figure 5 with 2 supplements
Role of each domain of Sec17 in zippering bypass fusion.
(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 …
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Figure 5—source data 1
Source data file (Excel) for Figure 5A–H,J and K.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Mean and standard deviations of fusion after 60 min from four independent assays as in Figure 5 are shown.
Figure 5—figure supplement 2
Sec18 does not simply promote fusion by contributing to the affinity of Sec17 for membranes that bear SNARE complexes.
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 …
Figure 6 with 1 supplement
Sec17, Sec18, and ATPγS restore fusion to Ypt7/R and Ypt7/Qa proteoliposomes, which were triply-crippled from completion of SNARE domain zippering by deletion of the +4 to +8 layers of the sQb and Qc SNAREs and by substitution of the apolar residues of the +4 to +8 layers of Qa, substituting Ala, Ser, or Gly for L238, M242, A245, L249, and A252.
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 …
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Figure 6—source data 1
Source data file (Excel) for Figure 6A–F.
- https://cdn.elifesciences.org/articles/67578/elife-67578-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Fusion assays between Ypt7/R- and Ypt7/Qa with Qa w.t. (black), Qa 5Ala mutant (green), Qa 5Gly mutant (blue), or Qa 5Ser mutant (red) as described in Figure 6.
(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.
Figure 7
Current working model.
Catalyzed tethering and SNARE assembly (A–C) is followed by HOPS displacement by Sec17 (C and D) and further assembly of Sec17 and Sec18 on the partially zippered SNARE complex, promoting completion …
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 | 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 |
