RECOVERY OF S-CEREVISIAE A-CELLS FROM G1-ARREST BY ALPHA-FACTOR PHEROMONE REQUIRES ENDOPEPTIDASE ACTION

Radioactive α factor is degraded to discrete biologically inactive fragments by the target a cells of S. cerevisiae, but not by α cells which make the pheromone. The pattern of cleavage products and sequence analysis of one fragment indicated that the first scission occurred between leucine 6 and lysine 7. The protease inhibitors tosyl-L-argininyl-methyl ester (TAME), tosyl-L-lysyl-chloromethylketone (TLCK) and N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin) markedly prolonged the period of G1 arrest in a cells exposed to α factor, while other standard protease inhibitors had little or no effect. The presence of TAME and leupeptin, or TLCK, reduced the rate of degradation of radioactively labeled α factor by a cells. Intact yeast cells have apparent esterase and amidase activities that are blocked by the same spectrum of inhibitors that potentiate α factor action. Purified α factor is a competitive inhibitor of these hydrolytic activities. The activities are present in yeast mutants which have greatly reduced levels of the three major vacuole-associated proteases (A, B and C) or which carry an ochre mutation in the major neutral protease (B). These observations indicate that the inactivation of α factor is due to endoproteolytic cleavage, that destruction of the pheromone is required to overcome its effects on growth and that degradation of the molecule may involve surface-bound endopeptidase(s). © 1979.