Feedback Regulation of Glucose Transporter Gene Transcription in Kluyveromyces lactis by Glucose Uptake

Abstract

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...

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

“…The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).…”

Transcriptomic Analysis of Extensive Changes in Metabolic Regulation in Kluyveromyces lactis Strains

“…5. Several studies have shown a correlation between respiration/fermentation and sugar transport (16,24,27,38), suggesting control of assimilation by fermentation due to a limitation of sugar uptake. It is notable that HGT1 is highly inducible, as the introduction of additional sugar transporter genes was shown to allow K. lactis to grow in the absence of respiration (16), and it is even more notable that the HGT1 gene is inactivated, suggesting that the mutation in HGT1 of strain B1 may compensate for the effects of the increased HGT1 gene expression and limit sugar uptake, thus preventing a too massive carbon uptake, which would be deleterious for the cell.…”

Isolation and characterization of theLGT1gene encoding a low-affinity glucose transporter fromTorulaspora delbrueckii

“…The former is only induced by high glucose, whereas induction of HXT3 expression by glucose is independent of sugar concentration. Regulation of LGT1 resembles more that of lowaffinity glucose transporters of K. lactis, KHT1 and RAG1, which show an enhanced expression with the external glucose concentration (Chen et al, 1992;Milkowski et al, 2001). We also analysed whether the Rgt1p and Mig1p consensus binding sites found in the promoter region of LGT1 were functional.…”

“…In spite of the phylogenetic proximity of this yeast to S. cerevisiae, the differences observed between the two species, demonstrate that the behaviour of T. delbrueckii cannot be directly inferred from that of S. cerevisiae. Furthermore, and specifically in what regards sugar transport, results from previous work have shown that other yeast species, also phylogenetically close to S. cerevisiae, show relevant differences at this level (Gonçalves et al, 2000;Milkowski et al, 2001). Therefore, an indepth investigation is required to gain insight into the function and regulation of T. delbrueckii sugar transporters.…”