Vanhalewyn M, Dumortier F, Debast G, Colombo S, Ma P, Winderickx J, Van Dijck P, Thevelein J M
Laboratorium voor Moleculaire Celbiologie, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Flanders, Belgium.
Mol Microbiol. 1999 Jul;33(2):363-76. doi: 10.1046/j.1365-2958.1999.01479.x.
In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.
在酿酒酵母中，向去阻遏细胞添加葡萄糖以及细胞内酸化会在1分钟内引发细胞内cAMP水平迅速升高。我们在几个相关的“野生型”实验室酵母菌株（如ENY.cat80 - 7A、CEN.PK2 - 1C）的遗传背景中发现了一种突变，该突变在很大程度上阻止了两种cAMP反应，我们将其命名为lcr1（意为缺乏cAMP反应）。随后的分析表明，lcr1与编码腺苷酸环化酶的CYR1/CDC35等位，并且它在第5627位存在A到T的替换。这对应于腺苷酸环化酶催化结构域末端附近的K1876M替换。将A5627T突变引入W303 - 1A野生型菌株的CYR1基因中，在很大程度上消除了葡萄糖和酸化诱导的cAMP信号传导，以及生长延迟期出现的cAMP短暂升高。因此，腺苷酸环化酶的赖氨酸1876对于体内cAMP反应至关重要。赖氨酸1876在粟酒裂殖酵母腺苷酸环化酶中保守。cyr1met1876（lcr1）菌株分离的质膜中依赖Mn2 +的腺苷酸环化酶活性与同基因野生型菌株相似，但依赖GTP/Mg2 +的活性大幅降低，这与体内通过腺苷酸环化酶的信号传导缺失一致。在cyr1met1876（lcr1）突变体中，葡萄糖诱导的海藻糖酶激活降低，海藻糖和糖原的动员以及应激抗性的丧失延迟。在葡萄糖上指数生长期间，对这些蛋白激酶A（PKA）靶点几乎没有影响，表明葡萄糖诱导的cAMP信号传导的重要性仅限于从糖异生/呼吸生长向发酵生长的转变。弱酸对生长的抑制作用降低，这与cyr1met1876（lcr1）突变阻止细胞内酸化对cAMP的影响一致。该突变部分抑制了RAS2val19和GPA2val132对几个PKA靶点的作用。这些结果证明了cyr1met1876（lcr1）突变在cAMP途径信号传导功能的上位性研究中的有用性。
