The hypocholesterolemic drug lovastatin (mevinolin) was found to be very effective in lowering the sterol levels of the wild-type yeast Saccharomyces cerevisiae. Lovastatin dramatically decreased the steryl ester content from 2.62 to 0.8 ,ug/mg (dry weight), whereas the free sterol content decreased only from 2.79 to 2.24 ,ug/mg (dry weight) when lovastatin was present in the medium at 10 ,ug/ml. At higher concentrations (100 ,ug/ml), lovastatin nearly abolished the accumulation of steryl esters and decreased the free sterol concentration to less than 1.3 ,ug/mg (dry weight). As a result of the lowered sterol levels, proportional amounts of exogenous sterol were taken up from the medium during aerobic, respiratory conditions. Nearly aUl of the exogenous sterol taken up was partitioned into the free sterol fraction. The inhibition of sterol esterification in the presence of lovastatin was dependent on heme synthesis. The result of these combined effects caused the MICs of three azole antifungal drugs (ketoconazole, clotrimazole, and miconazole) to be lowered from 6-to 32-fold when lovastatin was present in the medium at 10 Fg/ml.

There is an intimate association between sterol biosynthesis in yeast and aerobicity. Besides the requirement for molecular oxygen for the epoxidation of squalene, cytochrome hemoproteins are involved in demethylation and desaturation steps. Regulatory effects of hemes on sterol formation have been demonstrated using specifically defective mutants of yeast. Heme competency participates in a mechanism whereby wild-type cells are prevented from taking exogenous sterols from the growth media. The multiple interactions of hemes and sterols appear to be associated with the variously defined functions for sterols in the yeast cells.

A Saccharomyces cerevisiae sterol auxotroph, FY3 (a hem] erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 ,ug of sterol ml-', the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted. When cultured on 11 ,ug of sterol ml-' or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated. Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium. The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration. The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability. The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem] erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of 8-aminolevulinic acid to the medium allowed the sparking of FY3 (heml erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of 8-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 ,ug/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking ergosterol per ml.Evidence for differential utilization of sterols has been described in insects (3) and bacteria (5,6,17). Because of its ease of growth and facile genetic manipulations, Saccharomyces cerevisiae has been particularly valuable in studying the diverse functions of sterols. Using the sterol auxotrophs RD5-R and FY3, we demonstrated that the sterol requirement for growth could be fulfilled by cholestanol only when a very small concentration of ergosterol (10 ng/ml) was also present in the medium (23,25,26). We have systematically varied the combinations of sterols that were provided to the auxotrophs, and we proposed multiple roles for sterols in this organism under aerobic conditions. We have designated the cellular requirement that could be satisfied by cholestanol as the bulk function, and the high specificity function fulfilled by microamounts of ergosterol has been termed the sparking function. By feeding different sterols and stanols to RD5-R and FY3, we showed that most sterols and stanols could satisfy the bulk requirement. However, only those sterol derivatives that have a C-5,6 unsaturation or are capable of being desaturated at C-5 fulfill the sparking function (25).Other studies with S. cerevisiae have shown growth preferences for different sterols, depending on the cultural conditions. The sterol auxotroph GL7, grown on limiting amounts of ergosterol, has higher growth rates and yields in media additionally supplemented with cholesterol when compared with growth of the organism on either sterol alone (21). The necessity of the 24 P-methyl group on the sterol was proposed for S. cerevisiae when the wild-type organism is grown under anaerobic conditions in the presence of the synthetic sterol inhibitor 2, 3-iminosqualene (19, 20).Numerous sterols at nonlimiting concentrations (5 ,ug/ml or greater) appear to satisfy the sterol requirement in the presence (22,26) o...