Identification and Characterization of a cDNA Encoding a Dolichyl Pyrophosphate Phosphatase Located in the Endoplasmic Reticulum of Mammalian Cells

Abstract

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.