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Science 31 July 1998:
Vol. 281. no. 5377, pp. 700 - 702
DOI: 10.1126/science.281.5377.700
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Reports

Functional Reconstitution of Ypt7p GTPase and a Purified Vacuole SNARE Complex

Ken Sato, William Wickner *

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.
*   To whom correspondence should be addressed.

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