Functional reconstitution of ypt7p GTPase and a purified vacuole SNARE complex.
Membrane trafficking has heretofore been studied with intact organelles. Here, fusion-competent proteoliposomes were reconstituted from a yeast vacuole detergent extract. Homotypic vacuole fusion requires many membrane proteins, including the Ypt7p guanosine triphosphatase and a "SNARE complex" with Vam3p and Nyv1p. Proteoliposomes from extracts immunodepleted of either Vam3p or Ypt7p could not fuse, but vesicles reconstituted from a mixture of these depleted extracts had restored fusion activity. Purified preassembled vacuolar SNARE complex, when reconstituted with a SNARE-depleted extract, was fully functional for fusion. Thus, solubilized integral membrane components can be reconstituted for priming, docking, and fusion steps of organelle trafficking.
Sato
K
K
Dartmouth Medical School, Department of Biochemistry, Hanover, NH 03755-3844, USA.
Wickner
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins