Volume 272, Number 38, Issue of September 19, 1997 pp. 23540-23546
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

An Animal Cell Mutant with a Deficiency in Acyl/Alkyl-dihydroxyacetone-phosphate Reductase Activity
EFFECTS ON THE BIOSYNTHESIS OF ETHER-LINKED AND DIACYL GLYCEROLIPIDS

(Received for publication, March 21, 1997, and in revised form, June 25, 1997)

Paul F. James , Andrew C. Lake , Amiya K. Hajra ^¶ , Leslie K. Larkins ^¶ , Mitchell Robinson , F. Gregory Buchanan and Raphael A. Zoeller

From the  Department of Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, the ^¶ Department of Biological Chemistry and Mental Health Institute, University of Michigan, Ann Arbor, Michigan 48109, and the  Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA.K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO⁺ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-³H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with ³²P_i showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA.K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

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