Oxidative Stress-Induced Destruction of the Yeast C-Type Cyclin Ume3p Requires Phosphatidylinositol-Specific Phospholipase C and the 26S Proteasome

The yeast UME3 (SRB11/SSN3) gene encodes a C-type cyclin that represses the transcription of the HSP70 family member SSA1. To relieve this repression, Ume3p is rapidly destroyed in cells exposed to elevated temperatures. This report demonstrates that Ume3p levels are also reduced in cultures subjected to ethanol shock, oxidative stress, or carbon starvation or during growth on nonfermentable carbons. Of the three elements (RXXL, PEST, and cyclin box) previously shown to be required for heat-induced Ume3p destruction, only the cyclin box regulates Ume3p degradation in response to these stressors. The one exception observed was growth on nonfermentable carbons, which requires the PEST region. These findings indicate that yeast cells contain multiple, independent pathways that mediate stress-induced Ume3p degradation. Ume3p destruction in response to oxidative stress, but not to ethanol treatment, requires DOA4 and UMP1, two factors required for 26S proteasome activity. This result for the first time implicates ubiquitin-mediated proteolysis in C-type cyclin regulation. Similarly, the presence of a membrane stabilizer (sorbitol) or the loss of phosphatidylinositol-specific phospholipase C (PLC1) protects Ume3p from oxidative-stress-induced degradation. Finally, a ume3 null allele suppresses the growth defect of plc1 mutants in response to either elevated temperature or the presence of hydrogen peroxide. These results indicate that the growth defects observed in plc1 mutants are due to the failure to downregulate Ume3p. Taken together, these findings support a model in which Plc1p mediates an oxidative-stress signal from the plasma membrane that triggers Ume3p destruction through a Doa4p-dependent mechanism.

酵母UME3（SRB11/SSN3）基因编码一种C型细胞周期蛋白（C-type cyclin），该蛋白可抑制热休克蛋白70（HSP70）家族成员SSA1的转录。为解除该抑制作用，当细胞暴露于高温环境时，Ume3p会被快速降解。本研究表明，在受到乙醇冲击、氧化应激、碳源饥饿，或是在非发酵性碳源上生长的培养物中，Ume3p的蛋白水平同样会降低。在先前研究证实的、参与热诱导Ume3p降解的三个结构元件（RXXL基序、PEST序列与细胞周期蛋白盒（cyclin box））中，仅细胞周期蛋白盒可调控上述各类应激源诱导的Ume3p降解。唯一的例外是在非发酵性碳源上生长的情况，该过程依赖PEST序列。上述发现表明，酵母细胞存在多条独立的通路，可介导应激诱导的Ume3p降解。氧化应激诱导的Ume3p降解（而非乙醇处理诱导的该过程）需要DOA4与UMP1这两种26S蛋白酶体（26S proteasome）活性必需的调控因子。该结果首次将泛素介导的蛋白水解（ubiquitin-mediated proteolysis）与C型细胞周期蛋白的调控联系起来。类似地，膜稳定剂山梨醇（sorbitol）的添加，或是磷脂酰肌醇特异性磷脂酶C（PLC1）的缺失，均可使Ume3p免受氧化应激诱导的降解。最后，ume3无效等位基因（null allele）可抑制plc1突变体在高温或过氧化氢处理下出现的生长缺陷。上述结果表明，plc1突变体中观察到的生长缺陷，源于无法下调Ume3p的蛋白水平。综上，本研究结果支持如下模型：Plc1p介导来自质膜的氧化应激信号，并通过依赖Doa4p的通路触发Ume3p的降解。