Betz H, Weisner U
Eur J Biochem. 1976 Feb 2;62(1):65-76. doi: 10.1111/j.1432-1033.1976.tb10098.x.
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.
在酿酒酵母形成子囊孢子的过程中，至少60 - 70%的原有营养蛋白以相当恒定的速率被分解，直至成熟子囊出现。在相同条件下，源自同一菌株的非产孢单倍体中，蛋白质降解速率虽然最初与产孢细胞相当，但下降得更快。在孢子形成过程中不同时间合成的蛋白质，其降解速率与营养蛋白大致相同。通过差速离心从细胞匀浆中获得的所有组分中的营养蛋白，都观察到了相似的降解速率。转移到孢子形成培养基后，蛋白质分解被能量代谢的解偶联剂和抑制剂阻断，并且被环己酰亚胺部分抑制。在十二烷基硫酸钠存在下，对从营养细胞、分离的子囊和子囊孢子中提取的蛋白质进行聚丙烯酰胺凝胶电泳，结果显示子囊形成伴随着细胞蛋白质向较低分子量的转变。从几种测试的蛋白酶抑制剂中，只有甲苯磺酰 - p - 赖氨酸氯甲基酮略微降低了子囊形成的速率。在孢子形成过程中，蛋白酶A的总活性增加了两倍多，在转移到孢子形成培养基后18小时达到最大值。蛋白酶B的总活性在转移到孢子形成培养基后的最初几个小时内显著增加，之后在整个孢子形成过程中保持恒定。羧肽酶Y和蛋白酶B抑制剂的水平在孢子形成过程中没有显著变化。蛋白酶和蛋白酶B抑制剂存在于成熟的子囊孢子内。营养细胞和产孢细胞中的蛋白酶在相同离子强度下从DEAE - 葡聚糖凝胶上洗脱，并且它们对不同蛋白酶抑制剂的敏感性没有区别。以3H标记的变性酵母蛋白为底物，在产孢细胞中未检测到额外的蛋白水解活性。
