A mutant requiring both cholesterol and oleate for growth has been isolated from mutagenized Chinese hamster ovary cells. By comparison with wild-type cells, sterol and unsaturated fatty acid biosynthetic activities in the mutant cells grown in fetal calf serum medium appear to be nearly intact. However, whole-cell radioactive acetate, mevalonate, dihydrolanosterol, and stearate incorporation studies show that sterol synthesis from acetate, lanosterol demethylation, and fatty acid desaturation are defective in the mutant cells grown in delipidated serum medium. In vitro enzyme assays with crude cell extracts demonstrated that P-hydroxy-methylglutarylcoenzyme A reductase is not induced in the mutant. These experiments were substantiated by gas/liquid chromatographic analyses which showed the sterol content and the percentage unsaturated fatty acids in mutant cells to be drastically reduced when the cells are grown in delipidated serum medium. A spontaneous revertant exhibiting prototrophic growth in Ii id-free medium has been isolate from 50 X 100 mutant cells.A three defects in this revertant reverted back in parallel, which suggests that these three biosynthetic activities may be controlled by a common regulatory mechanism.Specific lipid-requiring mutants isolated from cultured animal cells (1-3) should serve as unique biological tools for studies of regulation of lipid metabolism and for membrane investigations. In this report, we describe the isolation of a Chinese hamster ovary (CHO) cell mutant (designated mutant clone no. 1) defective in the induction of cholesterol biosynthesis.Preliminary biochemical characterization indicates that this mutant is incapable of inducing its 13-hydroxy-fl-methylgluta- After 24 hr, the medium was replaced with 20 ml of F-12 + 0.5% dialyzed serum +1 mM DL-mevalonate + 0.013% methylcellulose (Fisher, 15 centipoises) (medium B) for 6 or 8 hr. Fresh 1 mM bromodeoxyuridine in saline was added to each flask (0.2 ml/flask). Cells were grown in this medium for 4347 hr. The medium was then drained off, the cells were washed twice with saline, and the flasks were exposed (bottom-side up) under a long-wavelength UV light source (UVSL-58 Mineralight, Ultraviolet Products, Inc.) at a distance of 5 cm for 15-20 min. Survivors were grown in medium A for 8 days, and the bromodeoxyuridine/light cycle (6) was repeated once more. Survivors were grown and cloned in medium A, and the colonies were tested for lipid auxotrophy by growing them in either 10% serum-supplemented or De-S medium. Potential auxotrophs were recloned before biochemical analyses. Spontaneous revertants were isolated by selecting survivors of 50 X 106 mutant clone no. 1 cells, 2 X 106 cells per 150-cm2 Coming flask, grown continuously in F-12 + 10% De-S medium with frequent medium changes for 30 days. Revertants were also cloned before biochemical analyses. Lipid Extraction Procedure. Cells were dissolved in 2 ml of 2 M NaOH as an aqueous slurry and transferred to graduated screw-capped conical extraction tubes (15...

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The relatively slow growth rate of the yeast mutant GL7, a sterol auxotroph, on medium containing cholesterol is markedly accelerated by supplementation with small amounts of ergosterol. Under these conditions (sterol synergism) cellular phospholipid synthesis is enhanced. We now find that one of the ergosterol-stimulated processes is the methylation of phosphatidylethanolamine to phosphatidylcholine. This is shown by comparing methyltransferase activities of membrane preparations derived from cells grown on either ergosterol, cholesterol, or the synergistic sterol pair. Incorporation of 32p from [y-32P]ATP into the yeast membranes is rapid and greater when ergosterol-grown cells rather than cholesterol-grown cells are the source of membranes.Under defined culture conditions, selected pairs of sterols promote substantially faster growth of the sterol auxotrophs Mycoplasma capricolum (1) and the yeast mutant GL7 (2) than do the corresponding sterols individually. We refer to this growth response, which is greater than additive, as sterol synergism. In M. capricolum a marked synergistic effect is seen with a 20:1 mixture of lanosterol and cholesterol, whereas for the yeast mutant a 3:1 mixture of cholesterol and ergosterol is the most effective pair. The optimal single sterol for supporting the growth of mycoplasma is cholesterol; for yeast it is ergosterol. In our view the synergistic growth responses we have observed with pairs of sterols are manifestations of dual or multiple functions of sterols in certain membrane systems (1-3). The conventional function, modulation of the membrane physical state, is assigned to the major component and distinguished from the role of the minor sterol component, which involves control of some metabolic membrane-associated processes. Evidence for such sterol-specific metabolic interventions in phospholipid biosynthesis has been reported. In M. capricolum cholesterol lowers the Km for oleic acid uptake and incorporation into phosphatidylglycerol severalfold (4). In the yeast mutant GL7, cellular ergosterol accelerates the rates of synthesis of all membrane phospholipids, as judged by incorporation of oleic acid (2).In exploratory experiments designed to identify ergosterolsensitive partial reactions of yeast phospholipid synthesis the first trial was negative. Fatty acyl-CoA synthetase activities in extracts of cells grown either on ergosterol or cholesterol were identical. Examining the transmethylation pathway for the synthesis of phosphatidylcholine (PtdCho) we observed, however, an ergosterol-specific promotion of enzyme activity. First, we found that yeast cells grown on ergosterol or ergosterol/cholesterol mixtures incorporate substantially more radioactivity from [methyl-3H]methionine into PtdCho and its partially methylated precursors than cells grown on cholesterol alone. This observation pointed to a role for the prototypic yeast sterol in the S-adenosylmethionine-mediated methylation of phosphatidylethanolamine (PtdEtn) to PtdCho. In accord with this expectatio...