Effect of ammonium ions on activity of hydrolytic enzymes during sporulation of yeast

Abstract

The addition of 10 mM ammonium sulfate to sporulation medium noncoordinately blocked the increases in protease C, protease B, ai-mannosidase, and 1,4amyloglucosidase activities which occur during normal sporulation of Saccharomyces cerevisiae, but had only a minor effect on the 10-fold increase in alkaline phosphatase activity.Sporulation of Saccharomyces cerevisiae has long been known to be inhibited by the addition of a nitrogen source to sporulation medium (13). NH4' blocks DNA, RNA, and protein syntheses (3,4,15), as well as the increase in protein (3) and glycogen degradation (5, 9) which occurs during normal sporulation. We have previously shown that NH4' has only a minimal effect on energy and glycolytic pathway metabolites in cells incubated in sporulation medium supplemented with NH4' (5). Therefore, we expected that the effects of NH4+ may be very specific. Since the differentiation during sporulation must necessitate significant amounts of intracellular macromolecular turnover, we examined the effect of NH4' on a number of hydrolytic enzymes which may be involved in intracellular turnover.S. cerevisiae strains SK-1 (9), AP-1-a/a, and AP-1-a/a (6) were used throughout this study. Yeast cells were sporulated in a manner similar to that used by Roth and Halvorson (16), as previously described (5). In experiments testing the effect of NH4', 10 mM (NH4)2SO4 was added to the sporulation medium. The minimum concentration of (NH4)2SO4 required to cause 95% inhibition of sporulation of SK-1 was 7.5 mM (unpublished data). Sporulation was monitored by phase-contrast microscopy as previously described (5).The cells were harvested and extracts were prepared as previously described (14). The addition of 2 mM phenylmethylsulfonyl fluoride had no effect on the levels of the non-proteolytic enzyme activities which were examined. Protease B activity was measured by the procedure of Juni and Heym (8), using Azocoll as substrate.

Comparative biochemistry of the proteinases of eucaryotic microorganisms.

“…A temporal relationship between levels of protein turnover and proteolytic enzymes can be seen in starving and differentiating cells of a number of species. In yeasts, the levels of proteinase A and B increase during sporulation as protein turn-324 NORTH rate of protein turnover and decreased proteinase activity (64,245). The vital role of the yeast proteinases in protein turnover can be assessed from the reduction of 30% and over 40o in protein degradation observed during sporulation of diploids lacking proteinase A (200) and proteinase B (358,377), respectively.…”

Sporulation in Hansenula wingei Is Induced by Nitrogen Starvation in Maltose-Containing Media

“…It is interesting to note that ammonium ion, the most powerful inhibitor of sporulation (17,18,20), is present at a low concentration in the plant. Ammonium ion plays a primary role in nitrogen metabolism in general by controlling the amino acid pool (13) but also appears to affect sporulation (secondary metabolism) specifically (19) by blocking the normal increases in proteases that occur in yeast cells after transfer to sporulation medium (15). Ammonium ion also inhibits RNA and protein synthesis during sporulation (9) as well as sporulation-specific glycogen degradation (11) and induction of glyoxylate enzymes during adaptation to acetate in Saccharomyces (12).…”