Characterization of KlGRR1 and SMS1 genes, two new elements of the glucose signaling pathway of Kluyveromyces lactis.

The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Deltasms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Deltasms1 mutation.

Connection between the Rag4 glucose sensor and the KlRgt1 repressor in Kluyveromyces lactis.

The RAG4 gene encodes for the sole transmembrane glucose sensor of Kluyveromyces lactis. A rag4 mutation leads to a fermentation-deficient phenotype (Rag- phenotype) and to a severe defect in the expression of the major glucose transporter gene RAG1. A recessive extragenic suppressor of the rag4 mutation has been identified. It encodes a protein (KlRgt1) 31% identical to the Saccharomyces cerevisiae Rgt1 regulator of the HXT genes (ScRgt1). The Klrgt1 null mutant displays abnormally high levels of RAG1 expression in the absence of glucose but still presents an induction of RAG1 expression in the presence of glucose. KlRgt1 is therefore only a repressor of RAG1. As described for ScRgt1, the KlRgt1 repressor function is controlled by phosphorylation in response to high glucose concentration and this phosphorylation is dependent on the sensor Rag4 and the casein kinase Rag8. However, contrary to that observed with ScRgt1, KlRgt1 is always bound to the RAG1 promoter. This article reveals that the key components of the glucose-signaling pathway are conserved between S. cerevisiae and K. lactis, but points out major differences in Rgt1 regulation and function that might reflect different carbon metabolism of these yeasts.

Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals.

Multidrug resistance in yeast results from overexpression of genes encoding drug efflux transporters owing to gain-of-function mutations in transcription factors regulating their expression. We have screened a library of synthetic compounds for modulators of drug resistance using the multidrug-resistant Saccharomyces cerevisiae pdr3-9 mutant strain. One of the compounds, 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT), displayed weak antifungal activity and strongly inhibited the growth of yeast cells in combination with subinhibitory concentrations of other antifungals with a different mode of action. Biological activity of CTBT was demonstrated in Saccharomyces, Kluyveromyces and Candida yeast species grown on solid and in liquid media. The chemosensitising effect of CTBT, manifested as increased antifungal activity of fluconazole, was demonstrated in yeast mutant strains with deleted genes encoding the major multidrug resistance transcription factors Yap1p, Pdr1p and Pdr3p as well as the drug efflux pumps Pdr5p and Snq2p in S. cerevisiae or their counterparts in Candida albicans and Candida glabrata, named Cdr1p and Mdr1p, respectively. Importantly, CTBT also increased the sensitivity to fluconazole in multidrug-resistant cells overexpressing the efflux pumps. Yeast cells grown in the presence of subinhibitory concentrations of CTBT exhibited an altered sterol composition and a slightly enhanced accumulation of Rhodamine 6G, which suggests that the plasma membrane plays a role in sensitisation. This novel chemosensitisation by CTBT that can overcome multidrug resistance in yeast may prove useful in combined treatment of infections caused by drug-resistant fungal pathogens.

Sck1 activator coordinates glucose transport and glycolysis and is controlled by Rag8 casein kinase I in Kluyveromyces lactis.

Casein kinases I (CKI) are ubiquitous in eukaryotic cells and are crucial factors for nutrient-signalling pathways in yeasts. In Kluyveromyces lactis, the KlRgt1 repressor represses the expression of the glucose transporter RAG1 gene in absence of glucose, but in response to glucose availability, Rag8 CKI cooperates with the Rag4 glucose sensor to inactivate KlRgt1. The SCK1 gene, a rag8 mutation suppressor, encodes a bHLH activator required for maximal expression of the RAG1 and glycolytic genes in the presence of glucose. We investigated further the function of Sck1 and its relationship to Rag8. We demonstrated that Sck1 regulates the RAG1 and glycolytic genes by directly binding to their promoter. We also found that SCK1 gene expression was induced by glucose and repressed by KlRgt1. In addition, we showed that (i) Sck1 was phosphorylated in vivo, (ii) Sck1 was phosphorylated in vitro by Rag8, and (iii) Sck1 was rapidly degraded in a rag8 mutant. We therefore suggest that Sck1 coordinates glucose import and glycolysis in K. lactis and that Rag8 controls this transcription factor by transcriptional and post-translational regulations.