The nuclear hexokinase 2 acts as an intracellular glucose sensor in Saccharomyces cerevisiae

Abstract

Glucose is not only a fuel that serves as a preferential substrate for energy yielding metabolism in the yeast Saccharomyces cerevisiae, but also functions as a signalling molecule that regulates the central pathways of carbohydrate metabolism. Under high glucose conditions, Mig1 is the main transcriptional factor responsible for the repression of genes needed for the utilization of alternative fermentable carbon sources, such as the SUC2 gene. It has been demonstrated that in several of the Mig1-regulated genes, this process requires the presence of the Hxk2 protein. In S. cerevisiae, Hxk2 is the predominant glucose-kinase in cells growing in high-glucose conditions and has dual functions. 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. Despite considerable effort in this field and recent progress in the last few years, little is known about the regulatory mechanism that controls nuclear Hxk2 association with the SUC2 promoter chromatin and how this association is necessary for SUC2 gene repression. Our data indicate that in the SUC2 promoter context, Hxk2 functions through a variety of structurally unrelated factors, mainly the DNA-binding Mig1 and Mig2 repressors and the regulatory Snf1 and Reg1 factors. Hxk2 sustains the repressor complex architecture maintaining transcriptional repression at the SUC2 gene. Using chromatin immunoprecipitation assays, we discovered that the Hxk2 in its open configuration, in low glucose conditions, leaves the repressor complex which induces its dissociation and promotes SUC2 gene expression. In high glucose conditions, Hxk2 adopts a close conformation that promotes Hxk2 binding to Mig1 protein and the reassembly of the SUC2 repressor complex. The formation and dissociation of the repression complex implicates the nucleocytoplasmic transport of the factors involved in this pathway. Our experiments suggest that the two regulatory factors Snf1 and Reg1, and the Snf4 and Gal83 proteins, in the same way as Hxk2, are transported into the nucleus by the classic import system Kap95/Kap60, and exported from the nucleus to the cytoplasm through the exportin Xpo1. Another mechanism that regulates the expression of the SUC2 gene is its subnuclear location. It has been demonstrated that the SUC2 gene is recruited to the nuclear pore complexes under high and low glucose conditions. Our data indicates that the nucleoporin Nup84 and Nup120 also form part of the repressor complex of the SUC2 promoter, and that Nup120 is involved in glucose repression. In addition, our results also suggest that Nup84 and Nup120 are also important in regulating Hxk2 recruitment to the SUC2 promoter. Additional findings highlight the possibility that Hxk2 constitutes an intracellular glucose sensor, which operates by changing its conformation in response to cytoplasmic glucose levels that regulate its interaction with Mig1 and thus its recruitment to the repressor complex of the SUC2 promoter. Thus, our data indicate that Hxk2 is more intimately involved in gene regulation than previously thought.