Protein degradation and proteinases during yeast sporulation

Abstract

During ascospore formation in Saccharomyces cerevisiae, at least 60-70% of the pre-existing vegetative protein was broken down at a rather constant rate until the time mature asci appeared. Under the same conditions in a non-sporulating haploid derived from the same strain the rate of protein degradation, although initially comparable to that of sporulating cells, decreased much more rapidly. Proteins synthesized at different times during sporulation had approximately the same degradation rates as the vegetative proteins. Similar rates of degradation were observed for the vegetative proteins in all fractions obtained from cell homogenates by differential centrifugation. Protein breakdown after transfer to sporulation medium was blocked by uncouplers and inhibitors of energy metabolism, and was partially inhibited by cycloheximide. Polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, of the proteins extracted from vegetative cells and from isolated asci and ascospores revealed that ascus formation was accompanied by a shift of the cellular proteins to a lower molecular weight. From several proteinase inhibitors tested, only tosyl-p-lysine chloromethylketone slightly reduced the rate of ascus formation. During sporulation the total activity of proteinase A increased more than twofold with a maximum at 18 h after transfer to sporulation medium. Total proteinase B activity showed a striking increase in the first hours after transfer to sporulation medium and after that remained constant throughout sporulation. The levels of carboxypeptidase Y and of the proteinase B inhibitor were not significantly altered during sporulation. The proteinases and the proteinase B inhibitor were present within the mature ascospore. The proteinases from both vegetative and sporulating cells were eluted with the same ionic strength from DEAE-Sephadex, and they were undistinguishable in their sensitivity to different proteinase inhibitors. No additional proteolytic activities could be detected in sporulating cells using 3H-labelled denatured yeast protein as a substrate.