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 fibroblastlike 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 [9,10-3 H]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 32 P 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 acyldihydroxyacetone 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.The formation of phosphatidic acid is required for the formation of diacyl glycerolipids in both bacterial and animal cells. In bacterial systems, this is initiated with the reduction of dihydroxyacetone phosphate (DHAP), 1 a product of glycolysis. The resulting sn-glycero-3-phosphate is acylated sequentially at the sn-1 and sn-2 positions to form phosphatidic acid. In animal cells, dihydroxyacetone phosphate (DHAP) can be acylated directly. In this "DHAP pathway," the ketone at the sn-2 position of the resulting sn-1-acyl-DHAP must be reduced, by acyl/alkyl-DHAP reductase (1), prior to further acylation. Purified acyl/ alkyl-DHAP reductase also catalyzes the reduction of the ether-linked sn-1-alkyl-DHAP (2), a reaction that is required in the synthesis of ether-linked glycerolipids, such as plasmenylethanolamine and glyceryl ether diesters (1). While the acylation of DHAP is accepted as the first step in ether-linked glycerolipids, the importance o...
