HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex.
Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Amy
A
Orr
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Hongki
H
Song
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Scott F
SF
Rusin
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.|Norris Cotton Cancer Center, Lebanon, NH 03766.
Arminja N
AN
Kettenbach
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 william.t.wickner@dartmouth.edu.
William
W
Wickner
D003593:Cytoplasm / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion; D008563:Membrane Lipids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D050600:SNARE Proteins / Q000378:metabolism* / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D008563:Membrane Lipids; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins