|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1997 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.