| I am using the unicellular eukaryote S. pombe as a model system in which to study processes that control anaphase onset. Since degradation of certain proteins is required for a proper exit from mitosis, I chose sensitivity to the protease inhibitor TPCK (N-Tosyl- sc L-Phenylalanine Chloromethyl Ketone) as a criterion by which to search for S. pombe mutants defective in the pathway for anaphase onset. To test this approach I examined thirty mitotic and cell cycle related mutants at their permissive temperatures for sensitivity to TPCK. Strains containing the mutations cut1-206, cut6-81, cdc15-136 and nuc2-663 were all found to be sensitive. The protein products of three of these genes, Cut1p, Cdc15p and Nuc2p, are likely to be involved in a proteolytic pathway that is now known to be important for anaphase onset. For example, nuc{dollar}sp{lcub}2+{rcub}{dollar} encodes a presumptive component of the Anaphase Promoting Complex, which is required for ubiquitin-dependent proteolysis of cyclin B.; Based on these findings I designed a novel genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at permissive temperature. We isolated three tsm (TPCK sensitive mitotic) strains, two of which are alleles of cut1{dollar}sp+.{dollar} One of these, cut1-tsm2, and a novel mitotic mutant tsm1-512 exhibit genetic interactions with the nuc2-663 mutation, supporting the idea that Cut1p and Tsm1p function at anaphase onset.; To study the role of Tsm1p I analyzed the phenotype of tsm1-512 at restrictive temperature. After shift to {dollar}36spcirc{dollar}C, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation. The chromosomes, however, often fail to segregate properly. My attempt to clone tsm1{dollar}sp+{dollar} by complementation rescue identified its multicopy suppressor sto1{dollar}sp+{dollar} (suppressor of tsm one). A deletion of sto1{dollar}sp+{dollar} leads to a mitotic arrest with no spindle. The predicted sequence of its protein product resembles that of a factor required for the folding of human {dollar}beta{dollar}-tubulin, suggesting that Sto1p is involved in microtubule function. Tsm1p and Sto1p are proposed to play roles in anaphase pathways related to microtubule function. |
