Colombo S, Ma P, Cauwenberg L, Winderickx J, Crauwels M, Teunissen A, Nauwelaers D, de Winde J H, Gorwa M F, Colavizza D, Thevelein J M
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Flanders, Belgium.
EMBO J. 1998 Jun 15;17(12):3326-41. doi: 10.1093/emboj/17.12.3326.
Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.
酿酒酵母中的腺苷酸环化酶活性依赖于Ras蛋白。向葡萄糖缺乏（去阻遏）的细胞中添加葡萄糖以及细胞内酸化都会在体内引发cAMP水平的升高。我们发现，细胞内酸化而非葡萄糖会导致Ras蛋白上的GTP/GDP比值升高，且这一过程不依赖于Cdc25和Sdc25。缺失GTP酶激活蛋白Ira1和Ira2，或表达RAS2(val19)等位基因，会导致GTP/GDP基础比值升高，并消除细胞内酸化诱导的升高。在ira1Delta ira2Delta菌株中，细胞内酸化仍会引发cAMP升高。在cAMP合成反馈抑制降低的菌株中，葡萄糖也不会导致GTP/GDP比值升高。进一步研究表明，cAPK对cAMP合成的反馈抑制作用独立于Ras上GTP/GDP比值的变化。葡萄糖刺激依赖于Gα蛋白Gpa2，其缺失会赋予与cAMP水平降低相关的典型表型：更高的耐热性、更高水平的海藻糖和糖原以及STRE控制基因的表达升高。然而，在葡萄糖上进行双相生长期间，这些特征的典型波动仍然存在。即使在蛋白激酶A减弱的菌株中，Ras2(val19)的过表达也会抑制酸化和葡萄糖诱导的cAMP升高。我们的结果表明，细胞内酸化至少通过激活Ras蛋白在体内刺激cAMP合成，而葡萄糖则通过Gpa2蛋白起作用。Ras2(val19)与腺苷酸环化酶的相互作用显然会阻止其被两种激动剂激活。
