Yeast Glycogen Synthase Kinase 3 Is Involved in Protein Degradation in Cooperation with Bul1, Bul2, and Rsp5

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs. The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3β and by an osmotic stabilizer. Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1. We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant. Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated. We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants. Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3. Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced. These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.

酿酒酵母（Saccharomyces cerevisiae）拥有MCK1、MDS1（RIM11）、MRK1以及YOL128c共四个编码糖原合成激酶3（glycogen synthase kinase 3，GSK-3）同源蛋白的基因。敲除这四个基因的GSK-3缺失突变体（gsk-3 null mutant）表现出温度敏感表型，该表型可通过哺乳动物GSK-3β的表达以及渗透压稳定剂得以恢复。bul1 bul2双缺失突变体同样可通过渗透压稳定剂恢复其温度敏感表型，且该突变体的温度敏感缺陷可通过多拷贝的MCK1得以抑制。本研究通过筛选可恢复mck1 mds1双缺失突变体温度敏感表型的rog突变体（即GSK-3回复突变体），发现其中两个突变体rog1和rog2同样能够恢复bul1 bul2双缺失突变体的温度敏感表型。已有研究表明，Bul1与Bul2可结合至Rsp5——一种属于HECT（即E6相关蛋白羧基末端同源域，homologous to E6-associated-protein carboxyl terminus）型泛素连接酶的蛋白——但尚未证实Bul1和Bul2参与蛋白质降解过程。本研究发现，在GSK-3缺失突变体以及bul1 bul2双缺失突变体中，Rog1的蛋白稳定性得以提升，但Rog2并无此现象。Rog1可与Rsp5直接结合，且二者的相互作用依赖于GSK-3。此外，在RSP5表达水平降低的npi1突变体中，Rog1的蛋白稳定性同样得以提升。上述结果表明，酿酒酵母GSK-3可通过与Bul1、Bul2以及Rsp5协同作用，调控Rog1的蛋白稳定性。