Biosynthesis of ergosterol in cell-free system of yeast

We previously proposed the occurrence of multiple pathways in the ergosterol biosynthesis of yeast, based on the results of examination of 14C-incorporation into sterols from L-[methyl-14C]methionine which was given to the intact cells of yeast. This led us to investigate the validity of the pathways by experiments with the cell-free system. Highly active cell-free extracts could be prepared by disruption of yeast cells with a Vibrogen Cell Mill in the presence of 0.1 mM dithiothreitol. This preparation catalyzed 14C-incorporation from [14C]methionine into ergosterol with a high yield. This preparation was found to be favorable for elucidation of ergosterol synthesis, since only a small amount of radioactivity was incorporated into fatty acid ester form of sterols which were reported to be inactive as a substrate for sterol synthesis reaction. Time course experiment of 14C-incorporation from [14C]methionine into various sterols under aerobic conditions showed that ergosta-8,24(28)-dien-3 beta-ol was a precursor for ergosta-7,24(28)-dien-3 beta-ol and that radioactivities were converted through ergosta-5,7,24(28)-trien-3 beta-ol and ergosta-5,7,22,24(28)-tetraen-3 beta-ol into ergosterol with time. In contrast, similar experiments under anaerobic conditions showed that ergosta-7,24(28)-dien-3 beta-ol accumulated and very little conversion of radioactivity into ergosterol occurred. In addition, the results indicated that oxygen was required for the introduction of double bond into 22 position as well as into 5 position. The results obtained with the cell-free system supported the validity of the proposal of multiple pathways of ergosterol synthesis in the intact cells.