Ahuatzi Deifilia, Herrero Pilar, de la Cera Tamara, Moreno Fernando
Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Campus del Cristo, 33006 Oviedo, Spain.
J Biol Chem. 2004 Apr 2;279(14):14440-6. doi: 10.1074/jbc.M313431200. Epub 2004 Jan 8.
Two major mediators of glucose repression in Saccharomyces cerevisiae are the proteins Mig1 and Hxk2. The mechanism of Hxk2-dependent glucose repression pathway is not well understood, but the Mig1-dependent part of the pathway has been elucidated in great detail. Here we report that Hxk2 has a glucose-regulated nuclear localization and that Mig1, a transcriptional repressor responsible for glucose repression of many genes, is required to sequester Hxk2 into the nucleus. Mig1 and Hxk2 interacted in vivo in a yeast two-hybrid assay and in vitro in immunoprecipitation and glutathione S-transferase pull-down experiments. We found that the Lys(6)-Met(15) decapeptide of Hxk2, which is necessary for nuclear localization of the protein, is also essential for interaction with the Mig1 protein. Our results also show that the Hxk2-Mig1 interaction is of physiological significance because both proteins have been found interacting together in a cluster with DNA fragments containing the MIG1 site of SUC2 promoter. We conclude that Hxk2 operates by interacting with Mig1 to generate a repressor complex located in the nucleus of S. cerevisiae during growth in glucose medium.
酿酒酵母中葡萄糖阻遏的两个主要介导因子是Mig1和Hxk2蛋白。Hxk2依赖性葡萄糖阻遏途径的机制尚未完全了解，但该途径中Mig1依赖性部分已得到详细阐明。在此我们报告，Hxk2具有葡萄糖调节的核定位，并且Mig1（一种负责许多基因葡萄糖阻遏的转录阻遏物）是将Hxk2隔离到细胞核中所必需的。在酵母双杂交试验中，Mig1和Hxk2在体内相互作用，在免疫沉淀和谷胱甘肽S-转移酶下拉实验中在体外相互作用。我们发现，Hxk2的Lys(6)-Met(15)十肽（该蛋白核定位所必需）对于与Mig1蛋白的相互作用也至关重要。我们的结果还表明，Hxk2-Mig1相互作用具有生理意义，因为已发现这两种蛋白在含有SUC2启动子MIG1位点的DNA片段的簇中一起相互作用。我们得出结论，在葡萄糖培养基中生长期间，Hxk2通过与Mig1相互作用来发挥作用，从而在酿酒酵母细胞核中产生一种阻遏复合物。
