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.
Dartmouth Medical School, Department of Biochemistry, Hanover, NH 03755-3844, USA.
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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