Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae

Abstract

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...

Cloning by Metabolic Interference in Yeast and Enzymatic Characterization of Arabidopsis thaliana Sterol Δ7-Reductase

“…Sterols that can satisfy this "sparking" effect must possess a double bond at C-5. Unsaturation at C-22 facilitates the sparking better than the C-7 unsaturation, and the 24␤-methyl group seems to be of low importance (19).…”

“…Nevertheless, the possibility to substitute ergosterol by its reduction product was a major concern since disruption of ergosterol biosynthesis is the classical mode of action of antifungal drugs like ketoconazole. In fact, the bulk (structural) function of the sterols can be fulfilled in yeast by different sterols such as cholestanol, cholesterol, lanosterol, or intermediates of sterol biosynthesis pathway, provided that some residual level of ergosterol be present (18,19). This is known as the "sparking effect," which is probably related to a cell cycle control mechanism in wild type strains (20).…”

Role of the non‐respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae

“…Nevertheless, the actual oxygen uptake linked to the haem pathway could not be checked in hem − mutants due to the pleiotropic effects of the hem mutations. 124,141,163 On the other hand, several data point to the strong contribution of de novo lipid synthesis to the CNR capacity of anaerobic cells. In such cells, the accumulation of both b5 and P450 cytochromes led some authors to conclude that sterol and unsaturated fatty acid (UFA) synthesis are the major CNR systems, but this could not be demonstrated.…”

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae

“…For this reason, the sterol C-5 desaturase was hypothesized to be essential in providing sparking sterol, and sterol-feeding experiments seemed to support the essential nature of this reaction. 149,216 Subsequently, the encoding yeast ERG3 gene was cloned and disruption of the gene resulted in a strain that retained viability under aerobic conditions. 5 The resolution of these contradictory conclusions lies in consideration of other markers in the strains used.…”