Publications

• The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae.

  Fernandez F., Rush J.S., Toke D.A., Han G.-S., Quinn J.E., Carman G.M., Choi J.-Y., Voelker D.R., Aebi M., Waechter C.J.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 276:41455-41464(2001) [PubMed] [EuropePMC]

• Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells.

  Rush J.S., Cho S.K., Jiang S., Hofmann S.L., Waechter C.J.

  The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphat ... >> More

  The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions. Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate. A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8Delta yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1Delta/dpp1Delta yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells. << Less

  J. Biol. Chem. 277:45226-45234(2002) [PubMed] [EuropePMC]