Asr1p在酿酒酵母细胞周期G2/M转变中的作用机制

The cell cycle is the basic process of cell life activities. Balance of polymerization and depolymerization of actin cytoskeleton involves in the regulation of G2/M transition before the bud formation and cytokinesis in Saccharomyces cerevisiae. However, there are few reports about how the actin cytoskeleton involves in the regulation of G2/M transition in the cell cycle of S.cerevisiae. In our previous work, we found that the cell cycle arrested at G2 stage and cells could not divide regularly after overexpression of the actin cytoskeleton regulational protein Asr1p. To prove the regulation roles and mechanisms of Asr1p in the G2/M transition of cell cycle, we first construct Asr1 gene mutant strains and test the regulation functions of Asr1p to Septin that plays important roles in the G2/M transition stage. Then demonstrate how Asr1p regulates Septin by the methods of yeast two-hybrid, pull down and Co-immunoprecipitaion and subcellular localization of protein. At last, check the phosphorylation level of targeted proteins located in the downstream of Septin. Combined with all the results above, we try to illustrate the roles of Asr1p play in the regulation of G2/M transition and initially reveal a new signaling pathway about G2/M transition regulated by actin cytoskeleton in S.cerevisiae.

细胞周期是细胞生命活动的基本过程。在酿酒酵母细胞周期G2/M转变子芽形成以及细胞分裂前，涉及肌动蛋白骨架聚合和解聚动态平衡的变化，但目前关于肌动蛋白骨架如何参与调节酿酒酵母细胞周期G2/M的转变知之甚少。申请者在前期研究中发现肌动蛋白骨架调节蛋白Asr1p过表达后使细胞周期停滞在G2期，细胞不能正常分裂。为了证明Asr1p在细胞周期G2/M转变中的调控作用及方式，本项研究首先构建Asr1 基因突变株，确认Asr1p对细胞周期G2/M转变中的功能蛋白Septin的调控作用。然后通过酵母双杂交、pull down、免疫共沉淀以及蛋白亚细胞定位实验证实Asr1p作用于Septin的方式。最后检测Septin下游靶标蛋白磷酸化水平的变化。通过本项目，阐明Asr1p在酿酒酵母细胞周期G2/M转变过程中的调控作用，初步揭示一条新的肌动蛋白骨架对细胞周期G2/M转变调控的信号通路。

细胞周期是细胞生命活动的基本过程。在酿酒酵母细胞周期G2/M 转变子芽形成以及细胞分裂前，涉及肌动蛋白骨架聚合和解聚动态平衡的变化，但目前关于肌动蛋白骨架如何参与调节酿酒酵母细胞周期G2/M 的转变机制不清楚。我们采用模式生物酿酒酵母为实验材料，首次证实酒精耐受蛋白Asr1p（Alcohol Sensitive Ring/PHD finger 1 protein）在细胞中的具体的生物学功能。①Asr1p参与肌动蛋白骨架动态平衡的调控；②Asr1p能够与保守的肌动蛋白骨架调控蛋白Coronin蛋白（Crn1p）相互作用，进而影响一系列的生物学过程：细胞周期、细胞大小、细胞生长、内吞等；③Asr1p对细胞周期的调控，是通过Crn1p与Septin家族成员Cdc11p的相互作用实现的，初步揭示了Asr1p通过Crn1p调控酿酒酵母细胞周期G2/M转变的信号通路。