Folia Microbiol 45 (6), 521-529 (2000) http: llwww .biomed. cas. ezlmbu/folia/ Sugar Repression in the Methylotrophic Yeast Hansenula polymorpha Studied by Using Hexokinase-Negative, Glucokinase-Negative and Double Kinase-Negative Mutants T. KRAMARENKO, H. KARP, A. JARVISTE,T. ALAMAE* Department of Genetics, Institute of Molecular and Cell Biology, Universityof Tartu. 51010 Tartu, Estonia fax + 372 7 420286 e-mail talamae@ebc.ee Received 14 June 2000 Revised version 27 December 2000 ABSTRACT. Two glucose-phosphorylating enzymes, a hexo- kinase phosphorylatingboth glucose and fructose, and a glu- cose-specificglucokinasewere electrophoreticaUy separated in the methylotrophic yeast Hansenulapolymorpha. Hexokinase- negative, glucokinase-negative and doublekinase-negative mu- tants were isolated in H. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was stu- died in different hexosekinase mutants. In the wild type and in mutants possessing either hexokinaseor glucokinase, glucose repressedthe synthesisof maltase, alcoholoxidaseand catalase. Glucose repression of alcohol oxidase and catalase was abo- lished in mutants lackingboth glucose-phosphorylating enzymes (i.e. in double kinase-negativemutants). Thus, glucoserepres- sion in H. polymorpha cells requiresa glucose-phosphorylating enzyme,eitherhexokinase or glucokinase.The presence of fruct- ose-phosphorylating hexokinase in the cell was specifically needed for fructoserepressionof alcohol oxidase, catalase and maltase. Hence,glucoseor fructosehas to be phosphorylated in order to cause repressionof the synthesis of these enzymesin H. polymorpha suggesting that sugar repression in this yeast therefore relieson the catalyticactivityof hexosekinases. In yeasts high concentrations of glucose in the medium repress the transcription of genes encoding enzymes responsible for the utilization of alternative carbon sources. The phenomenon is called glucose repression and its mechanisms have been thoroughly Studied in Saccharomyces cerevisiae using mostly MAL, SUC and GAL genes as a model (as review see Ronne 1995 and Gancedo 1998). In this yeast Migl protein is identified as the main transcriptional repressor binding to the promoters of glucose-repressed genes, and hexokinase PII protein is assumed to trigger the repression (Ronne 1995; Gancedo 1998; Klein et al. 1998). S. cerevisiae has three hexose kinases: the hexokinases PI and PII, which phosphorylate both glucose and fructose, and glucokinase, which is specific for glucose (as a review see Entian 1997). However, among the three glucose-phosphorylating enzymes only hexokinase PII seems to be involved in glucose repression under in vivo conditions. Mutants of S. cerevisiae with reduced glucose phosphorylation and defective glucose repression of invertase and maltase described in Zimmermann and Scheei (1977), revealed allelism with structural gene of hexokinase PII (Entian 1980). Study of mutants carrying different combinat- ions of null alleles of structural genes for hexose kinases also proved the absolute requirement for hexokinase PII in glucose repression (Ma and Botstein 1986; Walsh et al. 1991). However, if hexokinase PI was remo- ved in addition to hexokinase PII, glucose repression was further reduced, showing that hexokinase PI can also have some function in glucose repression (Rose et aL 1991). Despite extensive studies the mechanism of glucose repression triggering by hexokinase PII is still the matter of dispute. First, it has been suggested that elevated level of glucose 6-phosphate generated in the hexokinase reaction can be the triggerer of repression. This hypothesis is strongly supported by the inverse correlation between the catalytic activity of different hexokinase PII mutants of S. cerevisiae and the extent of glucose repression in these mutants (Ma et aL 1989; Rose et al. 1991). Hexokinase PII is a major glucose- phosphorylating enzyme in S. cerevisiae grown on glucose (Herrero et aL 1995) and should therefore be the main producer of glucose 6-phosphate under glucose repression conditions. According to the second hypo- thesis, hexokinase PII triggers glucose repression due to the presence of a specific regulatory domain in this protein. It is assumed that in the process of glucose binding and/or glucose phosphorylation a conformational change of the hexokinase protein takes place that triggers repression directly or through interaction with other proteins. This possibility is supported by the following data: (1) hexokinase PII mutants with unchan- ged catalytic activity but defective in glucose repression have been described (Entian and FrOhlich 1984); *Corresponding author.