title: The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae.
abstract: 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 P(i) 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.
authors.affiliation: Institut für Mikrobiologie, Eidgenössische Technische Hochschule Zentrum, Zürich CH-8092, Switzerland.
authors.forename: F
authors.initials: F
authors.lastname: Fernandez
authors.forename: J S
authors.initials: JS
authors.lastname: Rush
authors.forename: D A
authors.initials: DA
authors.lastname: Toke
authors.forename: G S
authors.initials: GS
authors.lastname: Han
authors.forename: J E
authors.initials: JE
authors.lastname: Quinn
authors.forename: G M
authors.initials: GM
authors.lastname: Carman
authors.forename: J Y
authors.initials: JY
authors.lastname: Choi
authors.forename: D R
authors.initials: DR
authors.lastname: Voelker
authors.forename: M
authors.initials: M
authors.lastname: Aebi
authors.forename: C J
authors.initials: CJ
authors.lastname: Waechter
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005656:Fungal Proteins / Q000235:genetics*; D008861:Microsomes / Q000201:enzymology; D009154:Mutation; D010641:Phenotype; D011755:Pyrophosphatases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera
chemical_list: C117838:CAX4 protein, S cerevisiae; D017931:DNA Primers; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011755:Pyrophosphatases; C023082:dolichyldiphosphatase