Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis.
Chinese hamster ovary (CHO) cells of the Lec9 recessive complementation group display a distinctive profile of resistance to a variety of toxic lectins. In addition, they accumulate cis-alpha-unsaturated polyprenol and use mainly polyprenol rather than dolichol to synthesize the glycosylated lipids used in asparagine-linked glycosylation of proteins. The primary defect in these cells is thought to result from a deficiency in polyprenol reductase activity. Three new mutants were isolated and determined to have qualitatively, although not quantitatively, similar lectin resistance profiles to Lec9 cells. Two of these mutants (AbrR and RicR) also contained polyprenol rather than dolichol. The lectin resistance profile of an independent mutant which accumulates polyprenol, F2A8, was also found to be qualitatively similar to the Lec9 pattern. The relationship among these mutants was analysed in more detail by construction of cell-cell hybrids. Lectin resistance profiles of the hybrids demonstrated that AbrR, RicR and F2A8 fell into the Lec9 complementation group. Analysis of prenols in the hybrids also showed that F2A8 was a member of the Lec9 group. Surprisingly, a significant fraction of the prenols found in Lec9 x Parent hybrids was polyprenol (up to 30% of the neutral fraction), whereas the prenols found in Parent x Parent hybrids were nearly exclusively dolichol (97% of the neutral lipid fraction). Therefore, reduction of polyprenol to dolichol appears to be a rate-limiting step in the synthesis of dolichol since hybrids with differing numbers of wild-type alleles can be biochemically distinguished.
Rosenwald
A G
AG
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205.
Stanley
P
P
McLachlan
K R
KR
Krag
S S
SS
D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D004286:Dolichols / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D005816:Genetic Complementation Test; D006822:Hybrid Cells / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D050356:Lipid Metabolism; D009154:Mutation; D010641:Phenotype; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*
D004286:Dolichols; D037102:Lectins; D011106:Polyisoprenyl Phosphates