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 budding yeast Kluyveromyces lactis glucose repression of genes involved in lactose and galactose metabolism is primarily mediated by LAC9 (or K1GAL4) the homologue of the well-known Saccharomyces cerevisiae transcriptional activator GAL4. Phenotypic difference in glucose repression existing between natural strains are due to differences in the LAC9 gene (Breunig, 1989, Mol.Gen.Genet. 261, 422-427). Comparison between the LAC9 alleles of repressible and non-repressible strains revealed that the phenotype is a result of differences in LAC9 gene expression. A two-basepair alteration in the LAC9 promoter region produces a promoter-down effect resulting in slightly reduced LAC9 protein levels under all growth conditions tested. In glucose/galactose medium any change in LAC9 expression drastically affects expression of LAC9 controlled genes e.g. those encoding beta-galactosidase or galactokinase revealing a strong dependence of the kinetics of induction on the LAC9 concentration. We propose that in tightly repressible strains the activator concentration drops below a critical threshold that is required for induction to occur. A model is presented to explain how small differences in activator levels are amplified to produce big changes in expression levels of metabolic genes.

. Cell. Biol. 7:991-997, 1987

The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes. Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned. A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype. The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4. Glucose repression is also eliminated by duplication of the LAC9-2 allele. The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator.