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J. Biol. Chem., Vol. 280, Issue 17, 16754-16762, April 29, 2005

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Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae^*

Vincent J. Starai, Naomi Thorngren, Rutilio A. Fratti, and William Wickner¶

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg²⁺ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca²⁺ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg²⁺ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca²⁺. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mM Mg²⁺, other free divalent ions are not needed. Reactions containing low Mg²⁺ concentrations are strongly inhibited by the rapid Ca²⁺ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca²⁺ or Mg²⁺, suggesting that Ca²⁺ does not perform a unique signaling function. When the need for Mg²⁺, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.

Received for publication, January 12, 2005 , and in revised form, February 22, 2005.

^* This work was supported in part 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.

Supported by a Damon Runyon Cancer Research Foundation fellowship (DRG-1837).

Supported by a Helen Hay Whitney Foundation fellowship.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755-3844. Tel.: 603-650-1701; Fax: 603-650-1353; E-mail: Bill.Wickner{at}Dartmouth.edu.

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