Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components.
Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.
Stroupe
Christopher
C
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Hickey
Christopher M
CM
Mima
Joji
J
Burfeind
Amy S
AS
Wickner
William
W
D000251:Adenosine Triphosphatases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D011510:Proteolipids / Q000378:metabolism / Q000648:ultrastructure; D050600:SNARE Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D011510:Proteolipids; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C020485:proteoliposomes; D006160:Guanosine Triphosphate; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins