Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.
Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.
Wang
Li
L
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Merz
Alexey J
AJ
Collins
Kevin M
KM
Wickner
William
W
D000199:Actins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins; D015003:Yeasts / Q000166:cytology / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C107706:VAM3 protein, S cerevisiae; C106736:VTI1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D020558:GTP Phosphohydrolases; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins