The Glucose-regulated Nuclear Localization of Hexokinase 2 in Saccharomyces cerevisiae Is Mig1-dependent

Abstract

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.The yeast Saccharomyces cerevisiae can use different carbon sources for growth, but evolution has selected mechanisms for the preferential and efficient utilization of glucose. In this yeast, glucose regulates carbon utilization mainly by the repression or activation of the transcription of numerous genes that encode enzymes implicated in carbon metabolism (1, 2). Although several of the genes implicated in the pathways that control glucose repression have been identified, a complete mechanistic picture of the process is not yet available. The Hxk2 and Mig1 proteins participate as important repressors in the glucose signaling pathway (3, 4).Hxk2 is the protein that initiates the intracellular metabolism of glucose by its phosphorylation at C-6, but in addition it plays a vital role in glucose repression (5). In hxk2 mutants repression of several genes by glucose is no longer operative (6, 7). If Hxk2 acts as a transcriptional repressor, it should be found in the cell nucleus at least under certain conditions. In fact, results obtained using different approaches have demonstrated that about 15% of the protein is localized in the nucleus (8,9) and that this localization is required for glucose repression of SUC2, HXK1, and GLK1 genes (9, 10). Moreover it has been shown that the nuclear Hxk2 is involved in the formation of specific DNA-protein complexes during glucose-dependent repression of these genes (9, 10).Mig1 is a C 2 H 2 zinc finger protein that binds to the motif WWWWWN(G/C)(C/T)GGGG in several promoters (11). Once bound to this MIG1 element, it recruits the Tup1-Cyc8 (Ssn6p) complex that represses gene transcription during growth in glucose (12). The activity of Mig1 is regulated by phosphorylation and subcellular localization. In high glucose, Mig1 is dephosphorylated by the Glc7-Reg1 protein phosphatase complex (13) and is located in the nucleus wher...

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

“…It is therefore possible that a situation similar to A. nidulans occurs in B. cinerea, but this is difficult to test without a double mutant. Nevertheless, the situation in Aspergillus and Botrytis is clearly different from that in yeast, in which Hxk2 directly interacts with the transcriptional repressor Mig1 (Ahuatzi et al, 2004). Thus, it remains to be elucidated how catabolite repression is mediated in B. cinerea.…”

“…In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi et al, 2004(Ahuatzi et al, , 2007.…”

“…In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi et al, 2004(Ahuatzi et al, , 2007.In filamentous fungi studied so far, a functional hexokinase and a functional glucokinase have been identified. In Aspergillus nidulans and Hypocrea jecorina, double mutants in both genes were unable to grow on either glucose or fructose (Flipphi et al, 2003;Hartl & Seiboth, 2005).…”

Hexokinase 2 Is an Intracellular Glucose Sensor of Yeast Cells That Maintains the Structure and Activity of Mig1 Protein Repressor Complex

“…Plasmids-GST fusion vectors pGEX/MIG1 and pGEX/ HXK2 for expression, respectively, of GST-Mig1 and GSTHxk2 were constructed as indicated (27). Plasmid pGEX-XPO1 for expression of GST-Xpo1 in E. coli was a gift from the C. N. Cole laboratory (49).…”

“…It is a glycolytic enzyme, essential for cell energy metabolism in the cytoplasm, but also acts as a regulator of gene transcription in the nucleus (26,27). To carry out their functions, Hxk2 has to shuttle in and out of the nucleus, but the protein is too large to translocate through the nuclear pore complex by diffusion.…”