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

本研究的目的是确定PAF（血小板活化因子）在出芽酵母中与细胞生长调节有关的可能功能。酿酒酵母不同生命周期细胞PAF产生和分布的变化研究酿酒酵母PAF产生和分布在细胞周期中的变化。细胞在G_1期产生的PAF最多，90%的PAF集中在细胞表面。我们发现，酵母在交配过程的早期产生PAF，以响应交配激素。利用STE6: URA3制备STE6基因干扰物（MAT a细胞），并与野生型细胞PAF的产生和分布进行比较。结果表明，由STE6基因编码的Ste6p不仅能转运脂肽交配激素a因子，还能转运脂质介质paf - 2。在培养基中加入PAFPAF调节细胞生长的机理，并对酵母细胞生长进行监测。PAF抑制细胞生长呈剂量依赖性。我们获得了PAF不敏感突变体，并检测了PAF对细胞内cAMP浓度、膜腺苷酸环化酶活性和磷脂酰肌醇（细胞IP_3）代谢的影响。结果表明，paf诱导的细胞生长抑制机制与RAS-cAMP通路和/或IP_3.3介导的信号转导有关。分离到paf敏感基因GPR1。GPR1基因编码的Gprlp不是PAF受体，而是定位于质膜的ag蛋白偶联受体，通过响应葡萄糖等胞外信号调控细胞cAMP水平。

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.