Molecular and genetical analysis of inducing factors for the G2/M transition in yeast.

在酿酒酵母中，Tom1基因编码泛素连接酶。Tom1突变体对温度敏感，受阻的细胞在细胞周期、mRNA输出、纺锤体和核仁结构以及胁迫反应的G2/M转变中存在缺陷。它们分别被鉴定为：Cyr1(腺苷酸环化酶)、sch9(A-激酶样蛋白)、mot1(转录抑制因子)、msi3(热休克蛋白)、cdc55(磷酸酶2A调节亚基)、zuo1(Z-DNA、tRNA结合蛋白)和kre6(参与葡聚糖合成)。由于cdc55和zuo1突变也被分离出来作为温度敏感型cdc20的反向突变，我们构建了cdc20和tom1的双重突变体，它不能在30ﾟC下生长。因此，泛素化M期抑制物和B型细胞周期蛋白的大复合体似乎与Tom1泛素连接酶有遗传关系。我们还寻找了Tom1泛素连接酶的候选底物采用双杂交系统。泛素和带有泛素样结构域的RAD23被分离出来，它们与hHZ结构域相互作用。鉴定出两个蛋白酶体亚基，这表明泛素途径和蛋白质降解机制是协同作用的。

The Tom1 gene encodes a ubiquitin ligase in Saccharomyces cerevisiae. The tom1 distuptant was temperature sensitive and the arrested cells were defective in G2/M transition in the cell cycle, mRNA export, spindle and nucleolar structures, as well as the stress response.We isolated pseudorevertants (tmr) of the tom1 mutant. They were identified as cyr1 (adenylate cyclase), sch9 (A-kinase-like), mot1 (transcriptional repressor), msi3 (heat shock protein), cdc55 (phosphatase 2A regulatory subunit), zuo1 (Z-DNA,tRNA-binding protein), and kre6 (involved in glucan synthesis). Since the STRE-dependent induction of the stress genes was known to be downdependent by the A-kinase pathway, it is reasonable to speculate that the tom1 mutant was not able to recover from heat shock.Since both the cdc55 and zuo1 mutations were also isolated as reversions of the temperature sensitive cdc20, we constructed the double mutant of cdc20 and tom1, which was not able to grow at 30ﾟC.Thus the large complex of APC (Anaphase Promoting Complex) which ubiquitinates the M-phase inhibitor and B-type cyclins, seems to be genetically related to Tom1-ubiquitin ligase.We also searched candidates for substrates of the Tom1-ubiquitin ligase, by using two-hybrid system. Ubiquitin and Rad23 carrying a ubiquitin-like domain were isolated which interacted with the hectdomain. Two proteasome subunits were identified, which suggested that the ubiquitin pathway and the protein degradation machinery were coordinately interacted.