Gancedo Juana M, Flores Carmen-Lisset, Gancedo Carlos
Department of Metabolism and Cell Signalling, Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM Arturo Duperier 4, E-28029 Madrid, Spain.
Biochim Biophys Acta. 2015 Jul;1850(7):1362-7. doi: 10.1016/j.bbagen.2015.03.006. Epub 2015 Mar 22.
A low level of glucose is required for maximal transcription of the SUC2 gene in Saccharomyces cerevisiae. Although the repressor Rgt1 binds the SUC2 promoter in gel-shift assays, it has been reported that Rgt1 has only minimal effects on SUC2 expression. Rgt1 acts together with Mth1 to repress the HXT genes encoding glucose transporters, and the release of Rgt1 from some HXT promoters requires cAMP-dependent protein kinase (PKA) activity.
The genes RGT1 and MTH1 have been disrupted and the SUC2 promoter modified in several S. cerevisiae backgrounds. Yeast cells were grown in different carbon sources in the presence or absence of 0.1 or 2% glucose, and invertase was assayed in whole cells.
Galactose, glycerol or ethanol hindered invertase induction by low glucose, but lactate did not. During growth in lactate, deletion of RGT1 or MTH1 caused a marked increase in invertase levels, and elimination of the Rgt1-binding site in the SUC2 promoter caused also invertase induction. PKA activity decreased invertase levels in cells growing in lactate, and increased them during growth in lactate+0.1% glucose.
The low level of expression of SUC2 in the absence of glucose is mainly due to repression by the Rgt1-Mth1 complex. Repression is dependent on PKA activity, but not on any specific Tpk isoenzyme.
The results show that previously overlooked regulatory elements, such as Rgt1 and Tpks, participate in the control of SUC2 expression in S.cerevisiae.
酿酒酵母中，SUC2基因的最大转录需要低水平的葡萄糖。尽管在凝胶迁移试验中阻遏蛋白Rgt1能结合SUC2启动子，但有报道称Rgt1对SUC2表达的影响极小。Rgt1与Mth1共同作用以抑制编码葡萄糖转运蛋白的HXT基因，并且Rgt1从一些HXT启动子上的释放需要环磷酸腺苷依赖性蛋白激酶（PKA）的活性。
在几种酿酒酵母背景中破坏了RGT1和MTH1基因，并对SUC2启动子进行了修饰。酵母细胞在存在或不存在0.1%或2%葡萄糖的情况下于不同碳源中生长，然后在全细胞中测定转化酶活性。
半乳糖、甘油或乙醇会阻碍低葡萄糖诱导的转化酶活性，但乳酸不会。在乳酸中生长期间，缺失RGT1或MTH1会导致转化酶水平显著增加，并且消除SUC2启动子中的Rgt1结合位点也会导致转化酶被诱导。PKA活性在乳酸中生长的细胞中降低了转化酶水平，而在乳酸+0.1%葡萄糖中生长期间则提高了转化酶水平。
在无葡萄糖时SUC2的低表达水平主要是由于Rgt1-Mth1复合物的抑制作用。抑制作用依赖于PKA活性，但不依赖于任何特定的Tpk同工酶。
结果表明，之前被忽视的调控元件，如Rgt1和Tpk，参与调控酿酒酵母中SUC2的表达。
