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J. Biol. Chem., Vol. 279, Issue 51, 53186-53195, December 17, 2004

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Diacylglycerol and Its Formation by Phospholipase C Regulate Rab- and SNARE-dependent Yeast Vacuole Fusion^*

Youngsoo Jun, Rutilio A. Fratti, and William Wickner

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

Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent homo-typic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequestered by a recombinant C1b domain. DAG underwent ATP-dependent turnover during vacuole fusion, but was replenished by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to DAG by PLC. The PLC inhibitors 3-nitrocoumarin and U73122 blocked vacuole fusion in vitro, whereas their inactive homologues did not. Plc1p is the only known PLC in yeast. Yeast cells lacking the PLC1 gene have many small vacuoles, indicating defects in protein trafficking to the vacuole or vacuole fusion, and purified Plc1p stimulates vacuole fusion. Docking-dependent Ca²⁺ efflux is absent in plc1 vacuoles and was restored only upon the addition of both Plc1p and the Vam7p SNARE. However, vacuoles purified from plc1 strains still retain PLC activity and significant 3-nitrocoumarin- and U73122-sensitive fusion, suggesting that there is another PLC in S. cerevisiae with an important role in vacuole fusion.

Received for publication, October 5, 2004

^* This work was supported in part by an NIGMS grant GM23377 from the 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.

Fellow of the Helen Hay Whitney Foundation.

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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