Basic and Applied Research on tie Roles of Ca^<2+>-Signal-mediated Cell-Cycle Regulation in Budding Yeast.

细胞周期调控的分析及其生理作用的研究为了了解酵母中Ca^2+信号介导的细胞周期调控的详细机制，我们分离了在这一过程中有缺陷的突变体（命名为scz），并对突变体进行了分子遗传学分析。(1)通过对scz 6突变的分析，我们发现该表型是由PKC 1基因突变引起的。我们进一步证明，C-激酶和A-激酶拮抗调节Cln 2，从而调节细胞生长和极化芽生长，这表明A-激酶和C-激酶途径之间的串扰。(2)通过对sczl 3突变的分析，我们发现了一个与ER腔蛋白N-糖基化有关的Alg 9突变，该突变与Ca^2+表型的抑制有关。我们发现蛋白质的N-糖基化对于维持Swel和Cln 2水平是重要的，我们还开发了一种检测抑制Ca^2+信号介导的细胞周期调节的生物活性化合物的方法，并筛选了各种来源的活性物质。对筛选出的两个小分子化合物的作用机理进行了研究。(1)Radicicol是Hsp 90的抑制剂，通过破坏Cdc 28/Clb的抑制性Idnase Swel的稳定性来抑制Ca^2+信号介导的细胞周期调节。(2)通过对NKH-7抗性突变株的分子遗传学分析，确定Tubl α-tubulin是NKH-7的作用靶点。基于NKH-7的生理作用和Tub I的突变位点，我们推测NKH-7可能通过与Tubl α-tubulin结合而破坏微管的稳定性。

Analyses of cell-cycle regulation and investigation of its physiological role For understanding detailed mechanism for the Ca^<2+>-signal mediated cell-cycle regulation in yeast, we isolated mutants (designated scz) defective in this process and performed molecular genetic analyses of the mutants.(1) By the analysis of scz6 mutation, we found that the phenotype is caused by a mutation of PKC1 gene. We further demonstrated that C-kinase and A-kinase antagonistically regulates Cln2, and thereby regulate cell growth and polarized bud growth, suggesting a crosstalk between A-kinase and C-kinase pathways.(2) By the analysis of sczl3 mutation, we found a mutation of Alg9 which is involved in the protein N-glycosylation in the ER lumen is responsible for suppression of the Ca^<2+> phenotype. We found that N-glycosylation of proteins is important for the maintenance of Swel and Cln2 levels.We also developed a procedure to detect bioactive compounds which inhibit Ca^<2+>-signal mediated cell-cycle regulation and screened for active substances from various sources. We investigated action mechanisms of two small-molecule compounds found by the screening.(1) Radicicol, inhibitor of Hsp90, inhibits Ca^<2+>-signal mediated cell-cycle regulation by destabilizing Swel, inhibitory Idnase of Cdc28/Clb.(2) By molecular genetic analysis of the mutants resistant to NKH-7, we identified Tubl α-tubulin as target of this compound. Based on its physiological effect and mutation site of Tub I, we suggested that NKH-7 destabilizes microtubules by binding to Tubl α-tubulin.