Nutrient Sensing through the Plasma Membrane of Eukaryotic Cells 265 Glucose sensing through the Hxk2-dependent signalling pathway F. Moreno 1 , D. Ahuatzi, A. Riera, C.A. Palomino and P. Herrero Departamento de Bioqu´ ımica y Biolog´ ıa Molecular, Universidad de Oviedo, 33006 Oviedo, Spain Abstract In this work, we describe the hexokinase 2 (Hxk2) signalling pathway within the yeast cell. Hxk2 and Mig1 are the two major factors of glucose repression in Saccharomyces cerevisiae. The functions of both proteins have been extensively studied but there is no information about possible interactions among them in the repression pathway. Our results demonstrate that Hxk2 interacts directly with Mig1 in vivo and in vitro and that the ten amino acids motif between K6 and M15 is required for their interaction. This interaction has been detected at the DNA level both in vivo by chromatin immunoprecipitation experiments and in vitro using purified proteins and a DNA fragment containing the MIG1 site of the SUC2 promoter. This demonstrates that the interaction is of physiological relevance. Our findings show that the main role of Hxk2 in the glucose signalling pathway is the interaction with Mig1 to generate a repressor complex located in the nucleus of S. cerevisiae. Introduction The ability of the yeast Saccharomyces cerevisiae to grow in different media is due to its capacity to sense and respond to changes in the availability of nutrients. Among sugars, glu- cose is probably the major signalling nutrient for S. cerevisiae as well as the carbon and energy source used preferentially. S. cerevisiae is able to detect the glucose levels of extracellular medium and to generate intracellular signals that result in an adequate cellular response to variations in the glucose medium composition. Many of these responses involve alter- ations in gene expression. The majority of these alterations occur at the level of mRNA transcription by the repression or activation of the transcription of many genes that encode enzymes implicated in carbon metabolism [1–3]. Present knowledge concerning factors required to transmit the glucose signal from the environment to the cell nucleus is mainly derived from studies with mutants affected in dif- ferent regulatory circuits governed by glucose [2]. In the last few years important advances in this field have been made [4–6]. These studies have characterized, in different detail, several signal transduction pathways that allow the yeast to perceive the level of glucose in the medium and to initiate the appropriate metabolic response (Figure 1). S. cerevisiae has membrane proteins that act as glucose re- ceptors. Glucose binds to these receptors and generates an intracellular signal. In the Rgt2/Snf3 pathway, these two pro- teins act as glucose receptors. The Rgt2 and Snf3 proteins resemble hexose transporters in structure but have long cyto- plasmic tails that are required for signal transduction [7]. Glucose binding to these transmembrane proteins initiates Key words: glucose, glucose repression, glucose sensing, hexokinase 2, Hxk2-dependent signalling pathway, Mig1. Abbreviations used: Gpr1, G-protein coupled receptor 1; GFP, green fluorescent protein; HA, haemagglutinin; Hxk2, hexokinase 2; PKA, protein kinase A. 1 To whom correspondence should be addressed (email fmoreno@uniovi.es). signals that activate a pathway that allows hexose transporter gene expression by repressing Rgt1 function [8]. An additional pathway that involves transcriptional changes in response to glucose is the stimulation of adenylyl cyclase and the increase in intracellular cyclic AMP. This path- way includes a G-protein coupled receptor (Gpr1) and two G proteins Gpa1 and 2, necessary for the glucose-specific in- crease in cAMP [9,10]. Finally, glucose activation of adenylyl cyclase leads to activation of the cAMP-dependent protein kinase A (PKA). Upon activation of PKA by cAMP the Rap1 transcription factor activity is increased inducing ex- pression of genes encoding ribosomal proteins and proteins required for glycolysis. In addition, PKA inactivates other transcription factors, such as Msn2 and Msn4, down-regu- lating the expression of STRE-controlled genes [11]. Once inside the cell, glucose activates another pathway involved in the repression of genes not needed during growth on glucose. In this pathway, both the Mig1 and the hexo- kinase 2 (Hxk2) proteins are necessary to generate the glucose repression signal. Thus, in hxk2 mutants repression of several genes by glucose is no longer operative even in the presence of the functional MIG1 gene. The mechanism by which Mig1 operates in this system is quite well known [12–14]. How- ever, the mechanism by which Hxk2 participates in the glu- cose signalling pathway, is almost unknown until now, in particular, the position of this factor in the signalling cascade and the interactions with the other factors of the cascade. Here we will briefly describe the Hxk2 signalling pathway in the S. cerevisiae cell, a protein that in addition to its classical meta- bolic role plays an important function in glucose signalling. Hxk2 interacts with the Mig1 protein and can enter the nucleus Hxk2, Mig1 and Med8 form part of the same protein com- plex that interacts with a regulatory element of the SUC2 C  2005 Biochemical Society