Distinct targeting and fusion functions of the PX and SNARE domains of yeast vacuolar Vam7p.
Regulated membrane fusion requires organelle tethering, enrichment of selected proteins and lipids at the fusion site, bilayer distortion, and lipid rearrangement. Yeast vacuole homotypic fusion requires regulatory lipids (ergosterol, diacylglycerol, and phosphoinositides), the Rab family GTPase Ypt7p, the multisubunit Ypt7p-effector complex HOPS (homotypic fusion and vacuole protein sorting), and four SNAREs. One SNARE, Vam7p, has an N-terminal PX domain which binds to phosphatidylinositol 3-phosphate (PI(3)P) and to HOPS and a C-terminal SNARE domain but no apolar membrane anchor. We have exploited an in vitro reaction of vacuole fusion to analyze the functions of each domain, removing the PX domain or mutating it to abolish its PI(3)P affinity. Lowering the PI(3)P affinity of the PX domain, or even deleting the PX domain, affects the fusion K(m) for Vam7p but not the maximal fusion rate. Fusion driven by the SNARE domain alone is strikingly enhanced by the PLC inhibitor U73122 through enhanced binding of Vam7p SNARE domain to vacuoles, and the further addition of Plc1p blocks this U73122 effect. The PX domain, through its affinities for phosphoinositides and HOPS, is thus exclusively required for enhancing the targeting of Vam7p rather than for execution of the Vam7p functions in HOPS.SNARE complex assembly and fusion.
Fratti
Rutilio A
RA
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Wickner
William
W
D004963:Estrenes / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011760:Pyrrolidinones / Q000494:pharmacology; D050767:Qc-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D004963:Estrenes; D046912:Multiprotein Complexes; D018129:Phosphatidylinositol Phosphates; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D050767:Qc-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae; C055525:phosphatidylinositol 3-phosphate; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D010738:Type C Phospholipases; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins