We studied 15-aza-24-methylene-8,14-cholestadiene-3,8-ol (15-azasterol) inhibition of Saccharomyces cerevisiae growth. Exposure to sublethal concentrations of this drug caused S. cerevisiae cells to undergo a transient period of inhibition at midlog phase. During growth inhibition the turbidity of each culture remained constant, as did the total cell number. Although the proportion of viable cells in cultures decreased from 90 to 12% during inhibition, methylene blue staining showed that less than 40% of the cells underwent metabolic inactivation. We monitored adenosine triphosphate levels throughout the inhibition cycle, and these levels followed kinetics identical to cell growth kinetics. After overcoming inhibition, cellular lipid extracts revealed the presence of a modified forn of 15-azasterol. It appeared that the yeast cells were able to overcome 15-aasterol inhibition by an inactivating transmethylation reaction involving S-adenosylmethionine.The drug 15-aza-24-methylene-8,14-cholestadiene-3,8-ol (15-azasterol) is a potent antimycotic compound that is produced by Geotrichum flavo-brunneum (7,13). Studies have shown that this compound inhibits sterol biosynthesis in fungi (8, 18) and in plants (15), causing the accumulation of A5,14 sterols. In Saccharomyces cerevisiae this inhibition occurs rapidiy after exposure to the drug (1), resulting in the synthesis of ergosta-8,14-3,B-ol (ignosterol) rather than ergosta-5,7,22-triene-3,8-ol (ergosterol), which is normally found in this yeast. At sublethal concentrations of 15-azasterol, S. cerevisiae shows no deviation from its normal growth pattern for the first few generations of growth. After this, cultures enter a period of growth inhibition, the length of which is dependent on the concentration of 15-azasterol added (8).In this study we examined the mechanism by which S. cerevisiae overcomes the growth inhibition mediated by 15-azasterol. Our experiments showed that S. cerevisiae is able to modify 15-azasterol to a non-inhibitory derivative by a transmethylation reaction involving S-adenosylmethionine.
MATERIALS AND MEETHODSFungal strains and culture conditions. Two of the organisms used in these experiments were S. cerevisiae strain S288C, which was obtained from the University of California at Berkeley, and S. cerevisiae t Technical paper 5882 from the Oregon Agricultural Experiment Station. strain A364a, which was obtained from George Sprague, University of Oregon. The 15-azasterol-resistant strain designated RG1 was derived by ethyl methane sulfonate mutagenesis of strain S288C (see below). G. flavo-brunneum strain NRRL 28804 was purchased from the American Type Culture Collection. Growth conditions for NRRL 28804 have been described previously (13). Yeast cells were grown in a liquid medium which contained 0.5% yeast extract, 1.0% tryptone, and 2.0% dextrose and was buffered with 0.1 M potassium succinate to pH 5.5 (designated YTDS medium). For a solid medium, 1.5% agar (Difco Laboratories) was added to this medium. Cells were cultured aerobically in 2...