Changing course: Glucose starvation drives nuclear accumulation of Hexokinase 2 in S. cerevisiae.

Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by hexokinases or glucokinases. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2, and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, yeast Hxk2 has been proposed to shuttle into the nucleus in glucose-replete conditions, where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve its role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 and requires an N-terminal nuclear localization sequence (NLS). We used high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. Countering previous yeast studies, we find that Hxk2 is largely excluded from the nucleus under glucose-replete conditions but is retained in the nucleus under glucose-limiting conditions. We find that the Hxk2 N-terminus does not contain an NLS but instead is necessary for nuclear exclusion and regulating multimerization. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Alanine substation at nearby lysine 13 affects dimerization and maintenance of nuclear exclusion in glucose-replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 regulates Hxk2 localization. RNAseq analyses of the yeast transcriptome dispels the idea that Hxk2 moonlights as a transcriptional regulator of glucose repression, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose-replete and limiting conditions. Our studies define a new model of cis- and trans-acting regulators of Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, which aligns well with the nuclear regulation of mammalian orthologs. Our results lay the foundation for future studies of Hxk2 nuclear activity.

Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics.

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.

Inwardly Rectifying Potassium Channel Kir2.1 and its "Kir-ious" Regulation by Protein Trafficking and Roles in Development and Disease.

Potassium (K+) homeostasis is tightly regulated for optimal cell and organismal health. Failure to control potassium balance results in disease, including cardiac arrythmias and developmental disorders. A family of inwardly rectifying potassium (Kir) channels helps cells maintain K+ levels. Encoded by KCNJ genes, Kir channels are comprised of a tetramer of Kir subunits, each of which contains two-transmembrane domains. The assembled Kir channel generates an ion selectivity filter for K+ at the monomer interface, which allows for K+ transit. Kir channels are found in many cell types and influence K+ homeostasis across the organism, impacting muscle, nerve and immune function. Kir2.1 is one of the best studied family members with well-defined roles in regulating heart rhythm, muscle contraction and bone development. Due to their expansive roles, it is not surprising that Kir mutations lead to disease, including cardiomyopathies, and neurological and metabolic disorders. Kir malfunction is linked to developmental defects, including underdeveloped skeletal systems and cerebellar abnormalities. Mutations in Kir2.1 cause the periodic paralysis, cardiac arrythmia, and developmental deficits associated with Andersen-Tawil Syndrome. Here we review the roles of Kir family member Kir2.1 in maintaining K+ balance with a specific focus on our understanding of Kir2.1 channel trafficking and emerging roles in development and disease. We provide a synopsis of the vital work focused on understanding the trafficking of Kir2.1 and its role in development.