Treitel M A, Kuchin S, Carlson M
Departments of Genetics and Development, Columbia University, New York, New York 10032, USA.
Mol Cell Biol. 1998 Nov;18(11):6273-80. doi: 10.1128/MCB.18.11.6273.
In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.
在以葡萄糖为碳源生长的细胞中，Mig1 DNA结合蛋白会将Ssn6-Tup1共抑制因子募集至酿酒酵母中受葡萄糖抑制的启动子区域。先前的研究表明，Mig1会因葡萄糖的刺激而发生差异磷酸化。在此，我们研究了Mig1在调节基因抑制中的作用以及Snf1蛋白激酶在调节Mig1功能中的作用。对snf1突变体中的Mig1蛋白进行免疫印迹分析表明，Snf1是Mig1磷酸化所必需的；此外，可解除葡萄糖对Snf1抑制作用的hxk2和reg1突变，相应地影响了Mig1的磷酸化。我们发现，Snf1和Mig1在双杂交系统中相互作用，并且也能从细胞提取物中共免疫沉淀，这表明这两种蛋白在体内相互作用。在Snf1的免疫复合物分析中，共沉淀的Mig1在Snf1依赖的反应中发生磷酸化。Mig1中四个假定的Snf1识别位点发生突变后，消除了Mig1在体内对葡萄糖响应的大部分差异磷酸化，并增强了与Snf1的双杂交相互作用。这些研究，连同先前的遗传学发现，表明Snf1蛋白激酶可响应葡萄糖调节Mig1的磷酸化。
