Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast

Abstract

Inhibition of sterol uptake in Saccharomyces cerevisiae sterol auxotroph FY3 (a hem) erg7 ura) by &-aminolevulinic acid (ALA) is dependent on the ability of the organism to synthesize heme from ALA. Sterol-depleted cells not exposed to ALA or strain PFY3 cells, with a double heme mutation, exposed to ALA did not exhibit inhibition of sterol uptake. Addition of ALA to sterol-depleted FY3 stimulated production of a high endogenous concentration of 2, 3-oxidosqualene (25.55 ,ug mg-' [dry weight]) at 24 h, whereas FY3 not exposed to ALA or PFY3 exposed to ALA did ikot accumulate 2,3-oxidosqualene. The high concentration of 2,3-oxidosqualene in FY3 with ALA decreased, and 2,3;22,23-dioxidosqualene increased to a very high level. The elevation of 2,3-oxidosqualene by ALA was correlated with a fivefold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34). The enhanced activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase was prevented by cycloheximide but not chloramphenicol and was dependent on a fermentative energy source. Inhibition of sterol uptake could not be attributed to 2,3-oxidosqualene or 2,3;22,23-dioxidosqualene but was due to a nonsaturating level of ergosterol produced as a consequence of heme competency through a leaky erg7 mutation.Biosynthesis of sterols and unsaturated fatty acids is an aerobic process in Saccharomyces cerevisiae (1). Hemoproteins are known to participate in several steps of the sterol biosynthetic pathway, including dehydrogenation and demethylation reactions (13,21). Thus, sterol biosynthesis is precluded in anaerobically cultured wild-type cells or in heme-deficient mutants cultured aerobically because of lack of the corresponding hemoproteins (2, 3, 12). Previous studies have indicated that when wild-type cells are grown aerobically, they are apparently impermeable to exogenous sterols (17, 27). Heme mutants, however, accumulate exogenous sterols from the medium under aerobic conditions (9, 26).We have shown recently that heme deficiency allowed sterol uptake in-a sterol auxotroph (15) and that cholesterol uptake was inversely proportional to the initial endogenous cellular concentration of cholesterol in a sterol-auxotrophic strain (16). That is, conditions that result in sterol-depleted cells or an endogenous sterol concentration below the saturation level allowed exogenous sterol uptake. Wild-type yeasts are capable of maintaining a free sterol saturation level throughout the growth cycle by using the steryl ester pool as a reserve source of free sterol (24) and thus do not take up exogenous sterol. However, in the heme tmutant FY3 (hemi erg7), which is additionally defective in 2,3-oxidosqualene cyclase, it has been found that, when 8-aminolevulinic acid (ALA) is supplied to allow heme synthesis, sterol uptake is inhibited (15).To understand the mechanism by which heme inhibits sterol uptake, a process known to be regulated only by the endogenous sterol concentration, we examined the effect of allowing heme biosynthesis on the activ...

Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

“…In fact, the pattern of the heme-mediated effects acted to buffer against changes in the total HMG-CoA reductase activity level. The lack of an effect on total cellular HMG-CoA reductase activity by heme depletion is in apparent conflict with an earlier report of heme depletion resulting in a substantial decrease in HMGCoA reductase activity (27). There are important differences between the experiments reported here and the earlier ones that may explain this discrepancy.…”

“…HMGCoA reductase activity has been reported to be higher in aerobically grown cells than in anaerobically grown cells (7,43). Studies with the yeast S. cerevisiae found HMG-CoA reductase activity to be lower in heme-deficient cells than in heme-sufficient cells, suggesting that heme acts as a positive regulator of HMG-CoA reductase (27). Since heme can act as a signal of oxygen levels in yeast cells, heme levels may mediate the effect of oxygen on the expression of the HMG-CoA reductase genes.…”

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

“…The attainment of heme competency in this strain has been shown to stimulate the activity of the sterol regulatory enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase at least fivefold. This derepression results in the biosynthesis and accumulation of 2,3-oxidosqualene, some of which can leak through the erg7 mutation to form ergosterol (13). Thus, we tested the ability of FY3 to spark on bulk cholestanol by utilizing ALA to biosynthesize endogenous ergosterol.…”

“…Heme incompetence was assayed by the inability to grow on YPD medium (1% yeast extract, 1% peptone, 2% glucose) without sterol and unsaturated fatty acids. Production of 2,3-oxidosqualene was determined by lipid extraction and gas chromatography (13). The presence of the sterol methyltransferase defect (erg6) was assayed by adding 0.1 pCi of [14C]methionine per ml to medium containing cholesta-5,7,22,24-tetraen-33-ol, unsaturated fatty acids, and 20 ,ug each of methionine and uracil per ml.…”

The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast

“…Their inclusion is associated with protection from toxic substances such as ethanol and sugar 59 . The steryl esters provide a pool of esterified 3␤-hydroxysterols, which can be hydrolysed if they are needed 60,97 . Different assumptions exist regarding the incorporation of these intracellular bound sterols into the membrane under conditions of oxygen limitation, but it seems likely that under anaerobic conditions cells with a large pool of neutral lipids show higher amounts of membrane bound sterols and thus increased survival capacity 76,77 .…”