Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

  1. Hongki Song
  2. Thomas L Torng
  3. Amy S Orr
  4. Axel T Brunger
  5. William T Wickner  Is a corresponding author
  1. Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, United States
  2. Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology Stanford University, United States
7 figures, 1 table and 1 additional file

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 …

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 …

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 …

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 …

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 …

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 …

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.

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 resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Saccharomyces cerevisiae)Nyv1Saccharomyces Genome DatabaseSGD:S000004083
Gene (Saccharomyces cerevisiae)Vam3Saccharomyces Genome DatabaseSGD:S000005632
Gene (Saccharomyces cerevisiae)Vti1Saccharomyces Genome DatabaseSGD:S000004810
Gene (Saccharomyces cerevisiae)Vam7Saccharomyces Genome DatabaseSGD:S000003180
Gene (Saccharomyces cerevisiae)Ypt7Saccharomyces Genome DatabaseSGD:S000004460
Gene (Saccharomyces cerevisiae)Sec17Saccharomyces Genome DatabaseSGD:S000000146
Gene (Saccharomyces cerevisiae)Sec18Saccharomyces Genome DatabaseSGD:S000000284
Gene (Saccharomyces cerevisiae)Vps33Saccharomyces Genome DatabaseSGD:S000004388
Gene (Saccharomyces cerevisiae)Vps39Saccharomyces Genome DatabaseSGD:S000002235
Gene (Saccharomyces cerevisiae)Vps41Saccharomyces Genome DatabaseSGD:S000002487
Gene (Saccharomyces cerevisiae)Vps16Saccharomyces Genome DatabaseSGD:S000005966
Gene (Saccharomyces cerevisiae)Vps11Saccharomyces Genome DatabaseSGD:S000004844
Gene (Saccharomyces cerevisiae)Vps18Saccharomyces Genome DatabaseSGD:S000004138
Peptide, recombinant proteinGST-R (Nyv1)PMID:18650938Purified from E. coli.
Peptide, recombinant proteinGST-Qa (Vam3)PMID:18650938Purified from E. coli.
Peptide, recombinant proteinGST-Qb (Vti1)PMID:18650938Purified from E. coli.
Peptide, recombinant proteinHis-R (Nyv1)PMID:22174414Purified from E. coli.
Peptide, recombinant proteinGST-sQa (soluble)PMID:28637767Purified from E. coli.
Peptide, recombinant proteinMBP-sQb (soluble)PMID:24088569Purified from E. coli.
Peptide, recombinant proteinGST-Qb (Vti1)3ΔThis studyPurified from E. coli.
Peptide, recombinant proteinGST-Qa (Vam3)3ΔThis studyPurified from E. coli.
Peptide, recombinant proteinQc (Vam7)PMID:17699614Purified from E. coli.
Peptide, recombinant proteinQc (Vam7)-tmPMID:23071309Purified from E. coli.
Peptide, recombinant proteinQc (Vam7) C208S, M250CThis studyPurified from E. coli.
Peptide, recombinant proteinQc (Vam7) C208S, A316CThis studyPurified from E. coli.
Peptide, recombinant proteinMBP-Cys-sQb (SNARE domain)PMID:28637767Purified from E. coli.
Peptide, recombinant proteinMBP-Cys-sQb (SNARE domain)PMID:28637767Purified from E. coli.
Peptide, recombinant proteinYpt7PMID:24088569Purified from E. coli.
Peptide, recombinant proteinYpt7-TMPMID:31235584Purified from E. coli.
Peptide, recombinant proteinSec17PMID:19414611Purified from E. coli.
Peptide, recombinant proteinSec17 FSMSPMID:28925353Purified from E. coli.
Peptide, recombinant proteinSec17 KEKEPMID:28925353Purified from E. coli.
Peptide, recombinant proteinSec17 LALAPMID:19414611Purified from E. coli.
Peptide, recombinant proteinGST-Sec17 6AtoNThis studyPurified from E. coli.
Peptide, recombinant proteinGST-Sec17-TMPMID:28718762Purified from E. coli.
Peptide, recombinant proteinGST-Sec17-TM FSMSPMID:28718762Purified from E. coli.
Peptide, recombinant proteinTEV proteasePMID:18007597Purified from E. coli.
Peptide, recombinant proteinHOPSPMID:18385512Purified from Saccharomyces cerevisiae.
AntibodyAnti-Vam3 (rabbit polyclonal)PMID:12566429Wickner lab stockWB: 1:2000
AntibodyAnti-Nyv1 (rabbit polyclonal)PMID:10385523Wickner lab stockWB: 0.65 μg/ml
AntibodyAnti-Vti1 (rabbit polyclonal)PMID:18007597Wickner lab stockWB: 0.47 μg/ml
AntibodyAnti-Vam7 (rabbit polyclonal)PMID:14734531Wickner lab stockWB: 0.1 μg/ml
AntibodyAnti-Vps16 (rabbit polyclonal)PMID:18007597Wickner lab stockWB: 1 μg/ml
AntibodyAnti-Vps33 (rabbit polyclonal)PMID:10944212Wickner lab stockInhibition assay: 1 μg
AntibodyAnti-Sec18 (rabbit polyclonal)PMID:11483507Wickner lab stockInhibition assay: 1 μg
Chemical compound, drugCy5-derivatized streptavidinSeraCare Life Sciences5270–0023
Chemical compound, drugBiotinylated PhycoEThermo Fisher Scientificp811
Chemical compound, drugStreptavidinThermo Fisher Scientific434302
Chemical compound, drug1,2-dilinoleoyl-sn-glycero-3-phosphocholineAvanti polar lipids850385
Chemical compound, drug1,2-dilinoleoyl-sn-glycero-3-phospho-L-serineAvanti polar lipids840040
Chemical compound, drug1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamineAvanti polar lipids850755
Chemical compound, drug1,2-dilinoleoyl-sn-glycero-3-phosphateAvanti polar lipids840885
Chemical compound, drugL-α-phosphatidylinositolAvanti polar lipids840044
Chemical compound, drug1,2-dipalmitoyl-sn-glycerolAvanti polar lipids800816
Chemical compound, drugErgosterolSigma45480
Chemical compound, drugPI(3)PEchelon BioscienceP-3016
Chemical compound, drugRhodamine DHPEInvitrogenL1392
Chemical compound, drugNBD-PEInvitrogenN360
Chemical compound,drugMarina-blueInvitrogenM12652
Software and algorithmsUN-SCAN-ITSilk Scientific
Chemical compound, drugAlexa Fluor 568 C5-maleimideThermo Fisher ScientificA20341
Chemical compound, drugOregon Green 488 MaleimideThermo Fisher ScientificO6034
Chemical compound, drugOregon Green 488 MaleimideThermo Fisher ScientificO6034
Chemical compound, drugPierce TCEP-HClThermo Fisher Scientific20490
Chemical compound, drugL-cysteineSigma-Aldrich30089

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