Glycolysis controls plasma membrane glucose sensors to promote glucose signaling in yeasts.

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.

Autoregulation of the Kluyveromyces lactis pyruvate decarboxylase gene KlPDC1 involves the regulatory gene RAG3.

In the yeast Kluyveromyces lactis, the pyruvate decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product--KlPdc1--through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.

The SWI/SNF KlSnf2 subunit controls the glucose signaling pathway to coordinate glycolysis and glucose transport in Kluyveromyces lactis.

In Kluyveromyces lactis, the expression of the major glucose permease gene RAG1 is controlled by extracellular glucose through a signaling cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 pathway. We have identified a key component of the K. lactis glucose signaling pathway by characterizing a new mutation, rag20-1, which impairs the regulation of RAG1 and hexokinase RAG5 genes by glucose. Functional complementation of the rag20-1 mutation identified the KlSNF2 gene, which encodes a protein 59% identical to S. cerevisiae Snf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds to RAG1 and RAG5 promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutive RAG1 gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism in K. lactis, notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway.

An efficient method to optimize Kluyveromyces lactis gene targeting.

Kluyveromyces lactis strains impaired in the nonhomologous end-joining pathway are relevant tools for the homologous integration of exogenous DNA into the genome, as in the mutant strains, close to 100% of the integrants are targeted to the homologous locus, compared with a few per cent for the wild-type recipient. Using a loxP-kanMX-loxP cassette together with a Cre-recombinase plasmid, a nej1∷loxP mutant strain suitable for multiple gene disruption has been constructed. Furthermore, using this strain, PCR-generated constructs with only 50 bp of homologous flanking sequences resulted in efficient exogenous DNA targeting.

KlHsl1 is a component of glycerol response pathways in the milk yeast Kluyveromyces lactis.

In Saccharomyces cerevisiae, HSL1 (NIK1) encodes a serine-threonine protein kinase involved in cell cycle control and morphogenesis. Deletion of its putative orthologue in Kluyveromyces lactis, KlHSL1, gives rise to sensitivity to the respiratory inhibitor antimycin A (AA). Resistance to AA on glucose (Rag+ phenotype) is associated with genes (RAG) required for glucose metabolism/glycolysis. To understand the relationship between RAG and KlHSL1, rag and Klhsl1Δ mutant strains were investigated. The analysis showed that all the mutants contained a phosphorylated form of Hog1 and displayed an inability to synthesize/accumulate glycerol as a compatible solute. In addition, rag mutants also showed alterations in both cell wall and membrane fatty acids. The pleiotropic defects of these strains indicate that a common pathway regulates glucose utilization and stress response mechanisms, suggesting impaired adaptation of the plasma membrane/cell wall during the respiratory-fermentative transition. KlHsl1 could be the link between these adaptive pathways and the morphogenetic checkpoint.

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.

Soil eukaryotic functional diversity, a metatranscriptomic approach.

To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

Mutations of the RAG3 gene encoding a regulator of fermentation in Kluyveromyces lactis are suppressed by a mutation of the transcription factor gene KlGCR1.

In Kluyveromyces lactis, Rag3 regulates both fermentative metabolism and thiamine biosynthesis. Regulation of fermentation is exerted at the level of transcription of KlPDC1. We have isolated and identified a mutation of the transcription factor KlGCR1, Klgcr1-1, which suppressed the fermentative-deficient phenotype associated with the RAG3 deletion. In the mutant, the transcription of KlPDC1 was restored. However, we found that the suppression was not specific to the RAG3 mutation, as the Klgcr1-1 mutation could also suppress the fermentative defect associated with mutation of Sck1, another regulator of glycolysis.

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.

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.

Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis.

We isolated a mutant, rag17, which is impaired in glucose induction of expression of the major glucose transporter gene RAG1. The RAG17 gene encodes a protein 87% identical to S. cerevisiae enolases (Eno1 and Eno2). The Kleno null mutant showed no detectable enolase enzymatic activity and has severe growth defects on glucose and gluconeogenic carbon sources, indicating that K. lactis has a single enolase gene. In addition to RAG1, the transcription of several glycolytic genes was also strongly reduced in the DeltaKleno mutant. Moreover, the defect in RAG1 expression was observed in other mutants of the glycolytic pathway (hexokinase and phosphoglycerate kinase). Therefore, it seems that the enolase and a functional glycolytic flux are necessary for induction of expression of the Rag1 glucose permease in K. lactis.

Regulation of glycolysis by casein kinase I (Rag8p) in Kluyveromyces lactis involves a DNA-binding protein, Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae.

The casein kinase I (Rag8p) of Kluyveromyces lactis has previously been shown to regulate the transcription of the low-affinity glucose transporter gene RAG1. To study this regulation, we have isolated multicopy suppressors of the rag8 mutation. One of them, SCK1 (suppressor of casein kinase), was characterised. The predicted product of the gene has a DNA-binding signature of the basic-helix-loop-helix type. It has an overall identity of 38% with Sgc1p (Tye7p) of Saccharomyces cerevisiae. The sck1 null mutant exhibited a Rag- phenotype (which indicates a reduced flux of glycolysis) that can be complemented by the SGC1 gene of S. cerevisiae. The level of transcription of several glycolytic genes, including RAG1, was reduced about twofold in glucose media in the sck1 null mutant. Moreover, in a rag8 mutant, the expression of SCK1 was strongly affected. Altogether, the results suggest that the regulation of glycolysis by casein kinase I involves, at least in part, Sck1p in K. lactis.