Vyas V K, Kuchin S, Carlson M
Integrated Program in Cellular Biology, Molecular Biology and Biophysical Studies, Columbia University, New York, NY 10032, USA.
Genetics. 2001 Jun;158(2):563-72. doi: 10.1093/genetics/158.2.563.
The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C(2)H(2) zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.
Snf1蛋白激酶对于酿酒酵母中葡萄糖抑制基因的转录至关重要。我们在双杂交系统中鉴定出Nrg2是一种与Snf1相互作用的蛋白。Nrg2是一种C(2)H(2)锌指蛋白,与Nrg1同源,Nrg1是葡萄糖和Snf1调节的STA1(葡糖淀粉酶)基因的阻遏物。在双杂交系统中,Snf1也与Nrg1相互作用,并从细胞提取物中与Nrg1和Nrg2共同免疫沉淀。与Nrg2的LexA融合蛋白可抑制含有LexA结合位点的启动子的转录,表明Nrg2也作为阻遏物发挥作用。与绿色荧光蛋白融合的Nrg1定位于细胞核,且这种定位不受碳源调节。最后,我们表明与Nrg1和Nrg2的VP16融合蛋白可使葡萄糖生长的细胞中SUC2低水平表达,并且我们提供证据表明Nrg1和Nrg2有助于对DOG2基因的葡萄糖抑制。这些结果表明Nrg1和Nrg2是Snf1激酶的直接或间接靶标,并在Snf1调节的一部分基因的葡萄糖抑制中发挥作用。