We have used the accumulated knowledge about the structure of the catalytic and regulatory domains of the Hxk2 protein to design a set of new mutations

We have used the accumulated knowledge about the structure of the catalytic and regulatory domains of the Hxk2 protein to design a set of new mutations. effect on the regulatory function of this protein. In the second, we analysed whether amino acids from Lys6to Met15of Hxk2 (Hxk2wrf) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In Fenoterol the present paper, we report that the Hxk2wcamutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2Mig1 repressor complex. We have demonstrated that Hxk2wcamaintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2wrfmutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2Mig1 repressor complex. The two mutants, Hxk2wcaand Hxk2wrfretain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2. Keywords:catalytic domain, glucose repression, glucose signalling, hexokinase 2, regulatory domain, yeast Abbreviations:ADH1, alcohol dehydrogenase 1; ChIP, chromatin immunoprecipitation; DTT, dithiothreitol; GAD, Gal4 activation domain; GBD, Gal4 DNA-binding domain Glk1, glucokinase 1; GST, glutathione transferase; HA, haemagglutinin; Hkx2, hexokinase 2;KanR, kanamycin-resistance; SD, synthetic dextrose; SG, synthetic galactose; wca, without catalytic activity; wrf, without regulatory function; YEPD, yeast extract/peptone/dextrose; YEPG, yeast extract/peptone/galactose == INTRODUCTION == There are three glucose-kinase isoenzymes inSaccharomyces cerevisiae[1]: isoenzymes 1 and 2 phosphorylate both aldo- and keto-sugars, whereas glucokinase is specific for aldo-hexoses. Isoenzyme 2 of hexokinase (Hxk2) is the predominant glucose-kinase inS. cerevisiaegrown on glucose media [2] and carries out at least two cellular functions. Classical structural studies of yeast Hxk2 reveal a well-defined catalytic domain that binds ATP and hexose (e.g. glucose), allowing transfer of a phosphoryl group from Fenoterol ATP to the C-6 of the sugar [35]. In addition to the well-known catalytic role of Hxk2, in the last few years, a new non-canonical function for this protein has been described. It has been suggested that the Hxk2 protein has special functions in transcriptional regulation [6]. Functional studies suggest that the main regulatory role of Hxk2 is produced by interaction with the transcriptional repressor Mig1 Fenoterol and the Snf1 protein kinase to generate a repressor complex in the nucleus [7,8]. Under high-glucose conditions, Hxk2 stabilizes the repressor complex, blocking Mig1 phosphorylation by Snf1 kinase [9]. Thus the complex is involved in the glucose-repression signalling of several Mig1-regulated genes. A determinant for the Hxk2Mig1 interaction has been characterized as an internal sequence between Lys6and Met15of the Hxk2 protein [10]. Moreover, it has been suggested that a fraction of the Hxk2 protein is sequestered to the nucleus by interacting with Mig1 through this amino acid region. Despite the large number of studies carried out to identify a correlation between catalytic activity Rabbit polyclonal to Argonaute4 and regulatory function in the Hxk2 protein, little is known about the existence of different domains in the protein that may control each activity. Both point [11] and null [12] mutations in theHXK2gene have been found that blocked glucose repression of certain genes. Since the glucose-phosphorylating activity in the corresponding extracts was reduced, the idea that there was a correlation between the glucose-phosphorylating activity of Hxk2 and glucose repression appeared a very attractive one [1,13]. However, this idea can be challenged if we take into account the following results: (i) when theGLK1(glucokinase 1) gene is overexpressed in a hxk1hxk2double-null mutant the transformed strains are still insensitive to glucose repression, even though a 3-fold increase in phosphorylating activity is achieved [1], (ii) glucose repression Fenoterol is not linearly reduced with decreasing kinase activity Fenoterol [14], and (iii) mutant alleles with low catalytic activity are still fully functional in glucose signalling [15]. These results suggest that sugar kinase activity and sugar signalling are mediated.