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

Tom1基因在酿酒酵母中编码一种泛素连接酶。tom1干扰物对温度敏感，阻滞细胞在细胞周期的G2/M转变、mRNA输出、纺锤体和核仁结构以及胁迫响应方面存在缺陷。我们分离了tom1突变体的伪回复性（tmr）。鉴定为cyr1（腺苷酸环化酶）、sch9 （a激酶样）、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）。由于已知应激基因的stret依赖性诱导是通过a激酶途径下调的，因此我们有理由推测tom1突变体无法从热休克中恢复过来。由于cdc55和zuo1突变也被分离出来作为温度敏感的cdc20的逆转，我们构建了cdc20和tom1的双突变体，该突变体在30ºC的温度下不能生长。因此，使m期抑制剂和b型细胞周期蛋白泛素化的APC（后期促进复合体）的大复合体似乎与tom1泛素连接酶有遗传关系。我们还利用双杂交系统寻找了tom1 -泛素连接酶的候选底物。分离到泛素和携带泛素样结构域的Rad23，它们与泛素结构域相互作用。两个蛋白酶体亚基的鉴定表明，泛素途径和蛋白质降解机制是协调相互作用的。

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.