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J. Biol. Chem., Vol. 282, Issue 20, 14861-14867, May 18, 2007

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Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification^*

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

SNARE proteins form bundles of four -helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q·1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7^Q283R, yet Vam7^Q283R is preferred by vacuoles with Nyv1^R191Q. Rotation of the position of the arginine in the 0-layer increases the K_m for Vam7p but does not affect the maximal rate of fusion. Vam7^Q283R forms stable 2Q·2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122 [GenBank] , perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.

Received for publication, February 1, 2007 , and in revised form, March 8, 2007.

^* This work was supported in part by a grant from NIGMS, National Institutes of Health. 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, Urbana-Champaign, IL 61801-3732.

² Current address: Dept. of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520-8024.

³ 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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