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Originally published In Press as doi:10.1074/jbc.M105544200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 44, 41455-41464, November 2, 2001
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The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae*

Fabiana FernandezDagger §, Jeffrey S. Rush§, David A. Toke||, Gil-soo Han||, Jeanette E. Quinn||, George M. Carman||, Jae-Yeon Choi**, Dennis R. Voelker**, Markus AebiDagger , and Charles J. WaechterDagger Dagger

From the  Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, the || Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08901-8520, the ** Department of Medicine, National Jewish Center, Denver, Colorado 80206, and the Dagger  Institut für Mikrobiologie, Eidgenössische Technische Hochschule Zentrum, Zürich CH-8092, Switzerland

Mutations in the CWH8 gene, which encodes an ER transmembrane protein with a phosphate binding pocket in Saccharomyces cerevisiae, result in a deficiency in dolichyl pyrophosphate (Dol-P-P)-linked oligosaccharide intermediate synthesis and protein N-glycosylation (van Berkel, M. A., Rieger, M., te Heesen, S., Ram, A. F., van den Ende, H., Aebi, M., and Klis, F. M. (1999) Glycobiology 9, 243-253). Genetic, enzymological, and topological approaches were taken to investigate the potential role of Cwh8p in Dol-P-P/Dol-P metabolism. Overexpression of Cwh8p in the yeast double mutant strain, lacking LPP1/DPP1, resulted in an impressive increase in Dol-P-P phosphatase activity, a relatively small increase in Dol-P phosphatase activity, but no change in phosphatidate (PA) phosphatase activity in microsomal fractions. The Dol-P-P phosphatase encoded by CWH8 is optimally active in the presence of 0.5% octyl glucoside and relatively unstable in Triton X-100, distinguishing this activity from the lipid phosphatases encoded by LPP1 and DPP1. Stoichiometric amounts of Pi and Dol-P are formed during the enzymatic reaction indicating that Cwh8p cleaves the anhydride linkage in Dol-P-P. Membrane fractions from Sf-9 cells expressing Cwh8p contained a 30-fold higher level of Dol-P-P phosphatase activity, a slight increase in Dol-P phosphatase activity, but no increase in PA phosphatase relative to controls. This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA. In accord with this enzymatic function, Dol-P-P accumulated in cells lacking the Dol-P-P phosphatase. Topological studies using different approaches indicate that Cwh8p is a transmembrane protein with a luminally oriented active site. The specificity, subcellular location, and topological orientation of this novel enzyme are consistent with a role in the re-utilization of the glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis after its release during protein N-glycosylation reactions.

* This work was supported by National Institutes of Health (NIH) Grant GM36065 (to C. J. W.), by NIH Grant GM28140 (to G. M. C.), by NIH Grant GM32453 (to D. R. V.), by Grant 3100-057082 from the Swiss National Science Foundation (to M. A.), and by a Post-doctoral Fellowship (to F. F.) by EMBO.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky, College of Medicine, Lexington, KY 40536. Tel.: 859-323-6352; Fax: 859-323-1037; E-mail: waechte@pop.uky.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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