Studies on regulation of cell growth of budding yeast by platelet-activating factor (PAF)

本研究的目的是明确PAF（platelet-ctivating factor）在芽殖酵母细胞中的可能功能及其与细胞生长的关系。1.酿酒酵母细胞在生命周期各阶段PAF产生和分布的变化我们研究了酿酒酵母细胞在生命周期各阶段PAF产生和分布的变化。G1期细胞产生的PAF最多，90%的PAF定位于细胞表面。同时，我们还发现，在交配过程的早期，酵母对交配激素有反应，产生PAF。利用ste 6：URA 3构建了STE 6基因破坏株（MAT a细胞），并与野生型细胞进行了PAF产生和分布的比较。结果表明，STE 6基因编码的Ste 6p不仅能转运脂肽类交配激素α-因子，还能转运脂质介质PAF。2. PAFPAF对酵母细胞生长的调控机制研究将PAFPAF添加到酵母细胞培养基中，观察酵母细胞的生长情况。PAF呈剂量依赖性抑制细胞生长。我们获得了对PAF不敏感的突变体，并研究了PAF对细胞内cAMP浓度、膜腺苷酸环化酶活性和磷脂酰肌醇（IP_3）代谢的影响。结果表明PAF抑制细胞生长的机制可能与RAS-cAMP途径和/或IP_3介导的信号转导有关。3. PAF敏感基因的克隆GPR 1基因编码的GPRlp不是PAF受体，而是一种定位于细胞膜上的G蛋白偶联受体，它对葡萄糖等细胞外信号的应答起调节细胞内cAMP水平的作用。

The purpose of this investigation was to define the possible function of PAF (platelet-ctivating factor) in budding yeast in relation to cell growth regulation.1.Changes in PAF Production and Distribution of S.cerevisiae cells in Various phase of Life CycleWe examined the changes of PAF production and its cellular distribution in S.cerevisiae during cell cycle. PAF production of cells was the highest at G_1 phase, and 90 % of PAF was localized in the cell surface. And, we found that the yeast produces PAF in early stage of mating process in response to the mating hormone. Furthermore, STE6 gene disruptant (MAT a cell) was made by using ste6 : URA3, and PAF production and distribution of the disruptant were compared with those of wild type cells. Results indicated that Ste6p encoded by STE6 gene, transport not only lipopeptide mating hormone a-factor, but also lipid mediator PAF.2.Mechanism of Regulation of Cell Growth by PAFPAF was added to culture medium and yeast cell growth was monitored. PAF inhibited cell growth dose-ependent manner. We obtained PAP-insensitive mutants, and examined the effects of PAF on the concentration of intracellular cAMP, activities of membrane adenylate cyclase and metabolism of phosphatidyl inositol (cellular IP_3). Results showed that mechanisms of PAF-induced inhibition of cell growth were related to RAS-cAMP pathway and/or the signal transduction mediated by IP_3.3.Cloning of PAF-ensitive GenePAF-sensitive gene, GPR1 was isolated. Gprlp, encoded by GPR1 gene was not PAF receptor, but it is aG-protein coupled receptor which localized at plasma membrane, and regulates the cellular cAMP level in response to the extracellular signal such as glucose.