Kübler E, Mösch H U, Rupp S, Lisanti M P
Institut für Mikrobiologie, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany.
J Biol Chem. 1997 Aug 15;272(33):20321-3. doi: 10.1074/jbc.272.33.20321.
The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.
小GTP结合蛋白Ras和异源三聚体G蛋白是真核细胞生长和发育的关键调节因子。在哺乳动物细胞中，Ras的功能是响应生长因子调节丝裂原活化蛋白激酶途径，而许多异源三聚体GTP结合蛋白α亚基则通过激素作用导致的腺苷酸环化酶调节cAMP水平。相比之下，在酿酒酵母中，是Ras1和Ras2蛋白调节腺苷酸环化酶。在酵母的两个G蛋白α亚基（GPA1和GPA2）中，只有GPA1得到了充分研究，并显示在信息素刺激下对丝裂原活化蛋白激酶途径起负调节作用。在本报告中，我们表明，编码另一个酵母G蛋白α亚基的GPA2基因缺失会导致假菌丝发育缺陷。此外，在没有Ras2p的情况下，GPA2基因对正常生长是必不可少的。这两种表型都可以通过缺失PDE2基因产物来挽救，PDE2基因产物通过切割使cAMP失活，这表明这些表型可归因于细胞内cAMP水平低。支持这一观点的是，向生长培养基中添加外源性cAMP也足以挽救GPA2缺失菌株的表型。综上所述，我们的结果直接表明，一个G蛋白α亚基可以通过cAMP依赖性机制调节酿酒酵母的生长和假菌丝发育。在这些酵母GPA2缺失菌株中异源表达哺乳动物G蛋白α亚基可为在体内无效环境中对哺乳动物G蛋白功能进行突变分析提供有价值的工具。
