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

细胞周期调控分析及其生理作用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，我们分离了该过程中存在缺陷的突变体（命名为scz），并对突变体进行了分子遗传学分析。（1）通过对scz6突变的分析，我们发现该表型是由PKC1基因突变引起的。我们进一步证明C-激酶和A-激酶拮抗地调节Cln2，从而调节细胞生长和极化芽生长，这表明A-激酶和C-激酶途径之间存在串扰。(2)通过对sczl3突变的分析，我们发现参与ER腔内蛋白质N-糖基化的Alg9突变负责抑制Ca^2表型。我们发现蛋白质的N-糖基化对于维持Swel和Cln2水平很重要。我们还开发了检测抑制Ca ^ 2 -信号介导的细胞周期调节的生物活性化合物的程序，并筛选了来自不同来源的活性物质。我们研究了筛选发现的两种小分子化合物的作用机制。(1)Radicicol，Hsp90的抑制剂，通过破坏Cdc28/Clb的抑制性Idnase Swel，抑制Ca^2信号介导的细胞周期调节。(2)通过对NKH-7耐药突变体的分子遗传学分析，我们确定Tubl α-tubulin作为该化合物的靶标。基于其生理效应和 Tub I 的突变位点，我们认为 NKH-7 通过与 Tubl α-微管蛋白结合来破坏微管的稳定性。

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.