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J. Biol. Chem., Vol. 282, Issue 17, 13133-13138, April 27, 2007

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Distinct Targeting and Fusion Functions of the PX and SNARE Domains of Yeast Vacuolar Vam7p^*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

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 [GenBank] through enhanced binding of Vam7p SNARE domain to vacuoles, and the further addition of Plc1p blocks this U73122 [GenBank] 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.

Received for publication, January 22, 2007 , and in revised form, February 12, 2007.

^* This work was supported by a grant from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation. Current address: Dept. of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801-3732.

² To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755. Tel.: 603-650-1701; Fax: 603-650-1353; E-mail: Bill.Wickner{at}dartmouth.edu.

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				V. J. Starai, C. M. Hickey, and W. Wickner HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion Mol. Biol. Cell, June 1, 2008; 19(6): 2500 - 2508. [Abstract] [Full Text] [PDF]