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J. Biol. Chem., Vol. 279, Issue 7, 5338-5345, February 13, 2004

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Vacuole Membrane Topography of the DPP1-encoded Diacylglycerol Pyrophosphate Phosphatase Catalytic Site from Saccharomyces cerevisiae^*

Gil-Soo Han, Celeste N. Johnston, and George M. Carman

From the Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901

The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the -phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane.

Received for publication, October 27, 2003 , and in revised form, November 18, 2003.

^* This work was supported in part by U. S. Public Health Service, National Institutes of Health Grant GM-28140. 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.

To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); E-mail: carman{at}aesop.rutgers.edu.

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