Triglyceride Synthesis by the Intracellular Level of Long-Chain Acyl Coenzyme A for Lipid Synthesis

Triglyceride and phospholipids are the main constituents of glycerolipids in eucaryotic cells. They are synthesized via a common precursor, phosphatidic acid, although their physiological roles are entirely different. Despite the biological importance of these two lipid classes, little is known about the control mechanism involved in their synthesis. We have shown previously that there are two functionally distinct long-chain acyl coenzyme A (CoA) synthetases in Candida lipolytica, a hydrocarbon-utilizing yeast (2, 6). Acyl-CoA synthetase I produces a long-chain acyl-CoA utilized solely for lipid synthesis, and acyl-CoA synthetase II produces an acyl-CoA destined exclusively for 3-oxidation (2, 3). When grown on oleic acid, C. lipolytica mutant cells defective in acyl-CoA synthetase I accumulated much less long-chain acyl-CoA for lipid synthesis than did the wild-type cells (3). Therefore, glycerolipid synthesis in mutant cells will diminish according to the underlying control mechanism. The results presented in this paper indicate that the synthesis of triglyceride is controlled by the level of long-chain acyl-CoA available for lipid synthesis, whereas the synthesis of phospholipids is hardly affected. C. lipolytica NRRL Y-6795 was used as the wild-type strain. Mutant strains L-5 and L-7, defective in acyl-CoA synthetase I, were derived from the wild-type strain (2). Strains RL71 and RL7-8 were spontaneous revertants obtained from strain L-7 (2). The basal medium consisted of 0.17% yeast nitrogen base without amino acids and ammonium sulfate (Difco Laboratories), 1% Brij 58, 0.25% KH2PO4, and 0.25% K2HPO4. The basal medium was supplemented with a carbon source and ammonium sulfate, as specified in the footnotes to Tables 1, 2, and 3. Cells were grown aerobically at 25°C. Glycerolipids synthesized in mutant cells defective in acyl-CoA synthetase I (L-5 and L-7) were compared with those synthesized in wildtype (Y-6795) and revertant (RL7-1 and RL7-8) cells. Cells were cultivated with ["4C]oleic acid as the sole carbon source for seven to eight generations to label the cellular components uniformly. Wild-type cells incorporated more than 40% of the cellular radioactivity into the total lipid fraction, whereas mutant cells incorporated only ca. 11% (Table 1). This difference in the total lipid fraction coincided well with the difference found with triglyceride but not with phospholipids. Since revertant cells synthesized triglyceride to an extent comparable to that found in wild-type cells, it was concluded that the defect in acyl-CoA synthetase I caused the decrease in the synthesis of triglyceride. These results suggest that the level of long-chain acylCoA for lipid synthesis controls the extent of triglyceride synthesis. The experiments presented in Table 2 were designed to determine whether this mechanism actually applies. In experiment 1, cells were grown on [14C]lauric acid, instead of oleic acid. Since acyl-CoA synthetase I hardly utilizes lauric acid (1), wild-type cells must be unable to accumulate a high level of long-chain acyl-CoA for lipid synthesis. These cells should synthesize only a small amount of triglyceride, assuming that the mechanism suggested above is valid. In fact, the extent to which triglyceride was synthesized in wild-type cells was small and similar to that observed in mutant cells grown on oleic acid (Table 2, experiment 1). Experiment 2 was carried out with [14C]glucose as the sole carbon source. When grown on glucose, wild-type cells exhibited a low level of long-chain acyl-CoA for lipid synthesis (3). Wild-type and mutant cells, regardless of the phenotype of acyl-CoA synthetase I, synthesized very small amounts of triglyceride (Table 2, experiment 2). Although these results provide strong support for the mechanism in question, confirmation should be made as to whether mutant cells are also capable