The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes. The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive. KHT2 is tandemly arranged with KHTI which is identical to the low-affinity transporter gene RAGI, except for the Cterminus. Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHTl and KHT2 generating the chimeric gene RAGI. Recombination between KHTl and KHT2 was also found in mutants of JA6 selected as 2-deoxyglucose resistant colonies. These mutants, like k h f l k h d double mutants were unable to grow on glucose when respiration was blocked (Rag-phenotype) and glucose repression was strongly reduced. khtl or kht2 single mutants of JA6 were Rag' but still an influence of the kht mutations on glucose repression was detectable. Repression was not affected in a Rag-mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux. The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake.

In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower K m (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant. Thus, the KHT genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous ␤-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.Most organisms have evolved sophisticated regulatory strategies to adapt their metabolism to the availability of nutrients. Substrate uptake is a first key function that is regulated. Signals that control substrate uptake depend on the nature and concentration of available nutrients and the nutritional state of the cell. Since substrate uptake feeds back on the nutritional state, a regulatory circuit exists, the components of which are only beginning to be understood even in such intensely studied pathways as glycolysis in Saccharomyces cerevisiae (see reference 20 for a recent review).S. cerevisiae cells are apparently able to sense the extracellular glucose concentration and transmit the signal over the membrane into the cytoplasm (20, 32). Two types of receptors have been proposed: hexose transporter-like receptors, which are involved in controlling hexose transporter (HXT) gene expression (30), and a G protein-coupled receptor that is required for the activation of the protein kinase A signaling pathway by cyclic AMP (22,32,36).Intracellularly, glucose-phosphorylating enzymes play an important role in glucose regulation. Whether these enzymes exert their influence on glucose regulation through their metabolic activity or whether they function as intracellular signaling molecules is still a controversial issue (13,15,19,33).A signaling function has clearly been established for the galactose-phosphorylating enzyme of the yeast Kluyveromyces lactis. This galactokinase (KlGal1p) is required to activate transcription of lactose and galactose metabolic genes (26). Upon binding of its substrates galactose and ATP, KlGal1...