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 Kleno 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.

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IN most strains of Kluyveromyces lactis, the glucose uptake system relies on two nonredundant glucose transporters: a low-affinity permease encoded by RAG1 (WéSOLOWSKI-LOUVEL et al. 1992a) and a high-affinity permease encoded by HGT1 (BILLARD et al. 1996). HGT1 is constitutively expressed (BILLARD et al. 1996); expression of RAG1 is activated in the presence of high concentrations of glucose (CHEN et al. 1992; WéSOLOWSKI-LOUVEL et al. 1992a). The Rag1 permease is necessary for supporting fermentative growth, which requires a high flow of substrate. In the absence of Rag1, the cell becomes respiration dependent for growth on high-glucose media. Thus, rag1 cells have the Rag^– phenotype: they cannot grow on 5% glucose in the presence of antimycin A, which blocks respiration (GOFFRINI et al. 1989; WéSOLOWSKI-LOUVEL et al. 1992b).

To date, studies of Rag^– mutants have identified three key components that are involved in the positive regulation of RAG1 expression: the glucose sensor Rag4 (BETINA et al. 2001), hexokinase Rag5 (PRIOR et al. 1993), and casein kinase I Rag8 (BLAISONNEAU et al. 1997).

In this report we present the characterization of another gene in K. lactis implicated in RAG1 regulation: RAG17 (KlENO) coding for enolase.

Yeast strains and growth conditions:

Yeast strains are described in Table 1. Yeast cells were grown at 28° in a complete YP medium containing 1% Bacto yeast extract, 1% Bactopeptone (Difco, Detroit), supplemented with either 2% glucose (YPG) or a specified carbon source. Minimal medium containing 0.7% yeast nitrogen base without amino acids (Difco) and 2% glucose was supplemented with auxotrophic requirements. The Rag phenotype was tested on 5% complete glucose medium supplemented with 5 µM antimycin A. For G418 medium, YPG plates were supplemented with geneticin (200 µg/ml; Life Technologies). 5-FOA medium was prepared according to BOEKE et al. (1984). The media for plates were solidified by the addition of 2% Bactoagar (Difco). Escherichia coli XL1-blue was used as a cloning host and DNA propagation and was grown in LB medium.

View this table: In this window In a new window   TABLE 1 Yeast strains

 
Genetics methods have been described previously (WéSOLOWSKI et al. 1982; GOFFRINI et al. 1989).

Yeast transformation:

Replicative transformation of K. lactis was performed by electroporation. For integrative transformation of K. lactis, the procedure described by DOHMEN et al. (1991) was followed. Replicative and integrative transformations of Saccharomyces cerevisiae were standard.

Construction of deletion strains:

One-step gene deletions using kanMX4 or HIS3 selection markers bearing PCR-generated long flanking homology (LFH-PCR; WACH 1996) was used to construct the mutant strains MWK3, MLK43, MLY702, and MLY703 (Table1). The primers used for the KlENO LFH-PCR synthesis were P5' KlENO (5'-CACGTTCACAATCCAGGCACC-3'), P5'L KlENO (5'-CCGTCGACCTGCAGCGTACGTGGCATGTTTTTTTG-3'), P3'L KlENO (5'-GCTCGAATTCATCGATGATATTTGACTGTCACCAAC-3'), and P3'KlENO (5'-AGCGAAGATAGCGTTGGAACC-3'). The primers used for the KlPGK LFH-PCR synthesis were P5' KlPGK (5'-ACGATCTCGTCCTAGTGGAAGC-3'), P5'L KlPGK (5'-GGGGATCCGTCGACCTGCAGCGTACGCATTTTTATTAATTCTTGATCG-3'), P3'L KlPGK (5'-CGAGCTCGAATTCATCGATGATATAAATGTAGGATCCATCATCCC-3'), and P3'KlPGK (5'-TACGATGAACCAGTGCACAAG-3'). The primers used for the ScENO2 LFH-PCR synthesis were P5' ScENO2 (5'-ATCCTACTCTTGCCGTTGCCATCC-3'), P5'L ScENO2 (5'-GGGGATCCGTCGACCTGCAGCGTACGCATTATTATTGTAT GTTATAGTA-3'), P3'L ScENO2 (5'-AAACGAGCTCGAATTCATCGATGATATAAAGTGCTTTTAACTAAGAATT-3'), and P3' ScENO2 (5'-GTACTCATAGAGGTAGGCTAGACC-3'). For all the P5'L and P3'L primers, the KanMX4 or HIS3MX6 regions are in boldface type. All correct integrations were verified by Southern blot or PCR.

In S. cerevisiae, the ScENO2 gene was first disrupted in the diploid strain MLY701 by using a kanMX4 or a HIS3MX6 disruption cassette, yielding MLY702 and MLY703 strains, respectively (Table 1). After meiotic analysis of these two diploid strains, the Sceno2 haploid strains MLY704 (Sceno2::kanMX4) and MLY708 (Sceno2::HIS3) were obtained. The double null mutant strain Sceno1 Sceno2 was then constructed by crossing the MLY708 strain with the Sceno1 strain Y07286 (Table 1), yielding the MLY713 diploid.

In K. lactis, the KlENO and KlPGK genes were disrupted in the MW270-7B strain using the corresponding kanMX4 disruption cassettes, yielding MWK3 and MLK43 strains, respectively.

Plasmid constructions:

The ENO2 gene of S. cerevisiae was first PCR amplified from MLY701 genomic DNA by using the Pfx polymerase (Invitrogen, San Diego) and the P5' ScENO2/P3' ScENO2 primers. After phosphorylation with T4 polynucleotide kinase (New England Biolabs, Beverly, MA), the ScENO2 PCR product was cloned into the CEN-URA3 vector pRS416 (SIKORSKI and HIETER 1989) linearized with SmaI, yielding pML180. This plasmid could complement the slow growth of the Sceno2 mutant (MLY708, Table 1) and thus contained a functional copy of ScENO2. The 2.9-kb XhoI-NotI fragment of pML180, containing ScENO2, was then cloned into the CEN TRP1 vector pRS414 (SIKORSKI and HIETER 1989) digested with XhoI and NotI, yielding pML190. The ScENO2 was also introduced into a K. lactis centromeric vector by cloning the XbaI-XhoI 2.9-kb fragment of pML180 between the XbaI and SalI sites of the CEN URA3 pCXJ18 vector (CHEN 1996), yielding pML187.

The K. lactis KlENO gene was cloned into a S. cerevisiae vector by subcloning the 5.5-kb BamHI fragment of pMW1 (see below) into the BamHI site of pRS414, yielding pML183.

Cloning and sequencing of RAG17/KlENO gene:

The RAG17 gene was cloned by in vivo complementation of rag17 mutation (strain MWK2) with a K. lactis genomic library made in the KCp491 vector (PRIOR et al. 1993). Of 5000 Ura⁺ transformants, 4 were found to be Rag⁺. The complementing plasmids extracted from these four transformants and amplified in E. coli showed that three of them carried the same plasmid, pMW1 containing a 7.5-kb insert (Figure 1), and one a plasmid with a 10-kb insert overlapping with a pMW1 insert.

View larger version (14K): In this window In a new window Download PPT slide   FIGURE 1.— (A) Restriction map of recombinant plasmid pMW1 and localization of the three ORFs (large arrows) identified by nucleotide sequences. Small arrows indicate the direction of DNA sequencing. (B) Restriction map of the RAG17/ KlENO locus. (C) Disruption cassette of the KlENO gene with kanMX4 marker (open box) to construct the MWK3 strain (Table 1). The solid boxes indicate genomic sequences (see MATERIALS AND METHODS).

 
A 2856-bp fragment containing the entire RAG17/KlENO gene was sequenced on both strands.

Preparation of yeast RNA and probes:

Total RNA was extracted from cells grown to an OD₆₀₀ of 2–3. Poly(A⁺)-enriched mRNA was obtained using mRNA Separator (CLONTECH, Palo Alto, CA). Several gene probes used were restriction fragments: RAG6 probe was a 1.3-kb EcoRI-SalI fragment (BIANCHI et al. 1996); HGT1 probe was a 1.75-kb EcoRI-HindIII fragment containing the HGT1 gene (BILLARD et al. 1996). Other probes were obtained by PCR amplification using either K. lactis genomic DNA or the cloned genes as template. The oligonucleotides used were: 0487 (5'-GGGGTCGTAGAATTGGT-3') and 0369 (5'-GACGTAACCGTAGTAGAAG-3') for RAG1; RAG2-up (5'-TGTACGTTGATGGTACCAACG-3') and RAG2-down (5'-CAAGATAGAACCAGTAGAGTA-3') for RAG2; p2E1/9 (5'-GCCATCTGTGCAGCATCAAA-3') and p2E1/5 (5'-GGGAAGAAGATCGAGTAGTG-3') for RAG4; 470 (5'-GTGCCAGCTAATTTGATGGA-3') and 471 (5'-AGCAGCCACCAATTGGATTG-3') for RAG5; PENO1 (5'-ACTGGCTGTCTGACTAGC-3') and EndoY (5'-GTCTTAGCACCGGCCAAGTC-3') for KlENO; SCK1-up (5'-GAACACCAACATGTTCGCTACTC-3') and SCK1-down (5'-GACAACGAACGCAGTATCTTCGC-3') for SCK1; GCR1-up (5'-CACCAGTAACATGATACGGTCC-3') and GCR1-down (5'-GACCACCATCAGATATACTGTTGCC-3') for KlGCR1; GCR2-B (5'-TCAGCGATTTCAACAGATAT-3') and GCR2-D (5'-CTCATTGATCTGTTCCATAG-3') for KlGCR2. In all cases, specific probes of K. lactis actin gene (KlACT), KlrRNA 18S gene (18S), or KlAAC gene were used in parallel as quantitative references. In the case of KlACT, the probe was a 900-bp EcoRI-HindIII fragment. 18S and KlAAC probes were amplified by PCR using the following oligonucleotides: PKl-18S f (5'-ATCCTGCCAGTAGTCATATGC-3') and PKl-18S r (5'-CCACAAGGAGTACAGGTTAGC-3') for rRNA 18S; P5'KlAAC (5'-AGATGAAATGATCAAGCAAGG-3') and P3'KlAAC (5'-CGTACATGGAGATAACACCGG-3') for KlAAC.

Northern blot hybridization was quantified by scanning with a Cyclone Phosphoimager (Packard, Meriden, CT).

Preparation of cell-free extract and enzyme assays:

Whole-yeast-cell extracts were prepared by glass-bead disruption of cells isolated from log-phase cultures grown in YP medium containing either 2% glucose or 2% glycerol. Enolase activity was assayed (CLIFTON et al. 1978) and normalized to protein concentration determined by the Bradford protein assay (Bio-Rad, Richmond, CA).

Isolation of the KlENO gene and deduced amino acid sequence of its product:

In K. lactis, gene replacement by homologous recombination can be accomplished, but at lower frequencies compared to S. cerevisiae. Usually, the gene disruption cassette recombines at ectopic sites in the genome. While attempting to construct a rag4::URA3 gene disruption (BETINA et al. 2001), we identified a Rag^– mutant that is not allelic to rag4, although RAG1 transcription is highly reduced in this mutant. The mutation is also not allelic to rag5 and rag8 mutations, both of which affect genes that positively regulate the transcription of the RAG1 gene (CHEN et al. 1992). We named the mutation rag17-1 (strain MWK1 in Table 1). The precise position of the URA3 insertion in the gene is not known.

RAG17 was isolated from a CEN-based K. lactis plasmid library by complementation of the Rag^– phenotype of the rag17-1 mutation (MATERIALS AND METHODS). The partial nucleotide sequence of the DNA fragment in the complementing plasmid (Figure 1 and MATERIALS AND METHODS) revealed the presence of three ORFs: one encodes a protein 40% identical to the Fmo of S. cerevisiae, a flavin-containing monooxygenase involved in protein folding (SUH et al. 1999); another encodes a protein highly similar to the enolases of S. cerevisiae, Eno1 and Eno2 (HOLLAND et al. 1981); the third encodes a protein 28% identical to Rax1 of S. cerevisiae, a protein implicated in bud site selection (CHEN et al. 2000). Because of the Rag^– phenotype of the mutation, the best candidate for RAG17 was the ORF that encodes the glycolytic enzyme enolase (KlENO). The predicted protein (437 amino acids) is 88 and 87% identical to ScEno2 and ScEno1 of S. cerevisiae, respectively, and 64% identical to human -enolase (Swiss-Prot accession P06733).

A PCR-based gene deletion cassette for RAG17/KlENO (Figure 1; Table 1; see MATERIALS AND METHODS for details) was used to disrupt the gene in the MW270-7B strain. Southern blot analysis confirmed the KlENO disruption in some G418^R transformants, such as MWK3 (data not shown). We also demonstrated that the KlENO locus is modified in the original rag17-1 mutant (MWK1), suggesting an ectopic integration of the rag4::URA3 gene disruption cassette in the KlENO gene. Like the rag17-1 mutation, the Kleno null mutation leads to a Rag^– phenotype. The allelism of Kleno with rag17-1 was confirmed by the absence of complementation of the Kleno null mutation (strain MWK3) by the rag17-1 mutation (MW352-2D) in a diploid constructed by crossing these two mutants. Thus, the cloned KlENO gene indeed corresponds to the RAG17 locus.

Growth phenotype of the Kleno mutant:

Growth of the Kleno null mutant is severely reduced on media containing either glucose or glycerol as the sole carbon source (Figure 2). In addition, the mutant strain is unable to grow on ethanol as carbon source. It is noteworthy that the Kleno mutant can grow slowly on complete glucose medium: it exhibits a 4.5-fold increase of doubling time as compared to wild-type cells (450 min vs. 110 min; data not shown). Better growth of mutant cells on complete medium is probably supported by other carbon sources (e.g., amino acids) in this medium.

View larger version (30K): In this window In a new window Download PPT slide   FIGURE 2.— Growth phenotype of the Kleno null mutant strain. The wild-type strain MW270-7B (KlENO), and the isogenic mutant strain MWK3 (Kleno) were streaked onto single colonies on 2% glucose, 2% glycerol, and 2% ethanol minimal plates. The photographs were taken after 3 days of incubation at 28°.

 

Enolase activity of Kleno mutant and KlENO expression:

No detectable enolase activity is present in the Kleno mutant, regardless of the substrate used (glycerol or glucose; Figure 3A). This strongly suggests that KlENO is the single enolase-encoding gene in K. lactis. Presence of the KlENO sequence in a single copy and absence of other related sequences in the genome was confirmed by low-stringency Southern blotting (data not shown). In contrast, in S. cerevisiae, which possesses two enolase genes, enolase activity is still detectable in Sceno1 or Sceno2 single mutants grown on glucose or on glycerol. It was not possible to assay enolase activity in the double-mutant Sceno1Sceno2 since it is inviable on glucose or glycerol media (see below).

View larger version (53K): In this window In a new window Download PPT slide   FIGURE 3.— (A) Enolase activity in wild-type and enolase mutant strains of K. lactis and S. cerevisiae. Enzyme activity was assayed as described in MATERIALS AND METHODS from cells grown on glucose or glycerol. Specific activities are expressed as micromoles of the product formed per minute and per milligram of proteins. S. cerevisae strains were BM61-1A (ScENO1 ScENO2), MLY704 (ScENO1 Sceno2), and Y07286 (Sceno1 ScENO2). K. lactis strains were MW270-7B (KlENO) and MWK3 (Kleno). (B) Northern blot analysis of KlENO mRNA. Each slot was loaded with 5–10 µg of total RNA and electrophoresed on a 1.2% agarose-formaldehyde gel. The probes used are described in MATERIALS AND METHODS. Lane 1, MW270-7B (KlENO) strain grown on 2% glycerol; lane 2, MW270-7B (KlENO) strain grown on 2% glucose; lane 3, MWK3 (Kleno) strain grown on 2% glycerol; lane 4, MWK3 (Kleno) strain grown on 2% glucose.

 
KlENO transcription was examined by a Northern blot analysis. The level of the KlENO transcript is slightly higher (twofold) when the cells are grown on 2% glucose (Figure 3B) than when they are cultivated on 2% glycerol. This result is consistent with the increased level of enolase activity detected in glucose-grown cells as compared to glycerol-grown cells (Figure 3A). In the null mutant, no transcript could be detected whatever the carbon source used. These results demonstrate that K. lactis possesses the single gene KlENO coding for an enolase and expressed under glycolytic as well as neoglucogenic conditions.

The KlENO gene complements the Sceno1 Sceno2 mutations of S. cerevisiae:

The nucleotide sequences of the two enolase-encoding genes of S. cerevisiae, ScENO1 and ScENO2, are >90% identical. The major difference between the two genes is in their 5' noncoding sequence. Therefore, they are expressed differently: the gluconeogenic gene ScENO1 is constitutively expressed (COHEN et al. 1987); the glycolytic gene ScENO2 is induced by glucose (MCALISTER and HOLLAND 1982; COHEN et al. 1986). The Sceno1 null mutation has no phenotype (MCALISTER and HOLLAND 1982); the Sceno2 mutant grows more slowly on glucose than does the wild-type strain (NIEDENTHAL et al. 1999). Meiotic analysis of the diploid MLY713 (ScENO2/Sceno2::HIS3 ScENO1 Sceno1::kanMX4) showed that no G418^R His⁺ spores are viable on YP medium containing either 2% glucose or 2% glycerol, but viable G418^R His⁺ spores could be obtained on YP medium containing 0.1% glucose + 2% ethanol. Under those conditions the Sceno1Sceno2 mutant probably obtains energy by respiration, which is derepressed at low concentrations of glucose.

To test if the KlENO gene can complement the growth defect of Sceno1Sceno2, we sporulated the MLY713 diploid (Table 1) carrying a CEN-URA3 plasmid containing the ScENO2 gene. A meiotic segregant of a complete tetrad, MLY719 (Ura⁺ His⁺ G418^R Trp^–), was transformed in parallel with a CEN-TRP1 plasmid carrying either KlENO or ScENO2 (Table 1). In both cases the transformants were able to grow on 5-FOA medium, which counterselects for Ura⁺ cells (Figure 4). As a control, the same strain was transformed with the empty pRS414 plasmid and found unable to lose the plasmid carrying ScENO2 (pML180; i.e., this strain cannot grow on 5-FOA medium). We conclude that KlENO restores viability to the Sceno1Sceno2 mutant of S. cerevisiae. Thus, KlENO is a functional homolog of the ENO genes of S. cerevisiae. The reciprocal heterologous complementation was confirmed: ScENO2, cloned in a K. lactis centromeric vector (pML187; see MATERIALS AND METHODS), complements the Rag^– phenotype of the Kleno mutant (data not shown).

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 4.— Complementation of the Sceno1 Sceno2 double mutation of S. cerevisiae by the KlENO gene. The Sceno1Sceno2 cells (strain MLY719, Table 1), rescued with the pML180 plasmid (ScENO2 gene cloned in the URA3 vector pRS416), were transformed with TRP1 vector pRS414 (empty vector) or with the ScENO2 and KlENO genes individually carried by pRS414. Transformants were grown in parallel on uracil-less, tryptophan-less, and 5-FOA medium. The plates were incubated for 3 days at 28° before the photographs were taken.

 

KlENO is required for expression of genes encoding glucose permeases and glycolytic genes and their regulators:

Northern blot analysis presented in Figure 5 shows that the disruption of KlENO results in a severe reduction of transcript levels of both glucose transporter genes, RAG1 and HGT1. However, the transcription of HGT1 was less affected than that of RAG1. The transcription of the hexokinase (RAG5, KlHXK) and pyruvate decarboxylase gene (RAG6, KlPDC) is also impaired in the mutant. In contrast, the phosphoglucose isomerase gene (RAG2, KlPGI) is not affected (Figure 5).

View larger version (37K): In this window In a new window Download PPT slide   FIGURE 5.— Effect of the disruption of KlENO on the transcription of glucose-transporter and glycolytic genes. Approximately 5–10 µg of total RNA extracted from cells grown on 2% glucose was loaded in each slot. Strains used were MW270-7B (KlENO), MWK3 (Kleno1::kanMX4), and MWK1 (carrying the original Kleno::URA3 mutation) (Table 1). Electrophoresis conditions were as in Figure 3. The probes used to detect RAG1, HGT1, RAG5 (KlHXK coding for hexokinase), RAG2 (KlPGI coding for phosphoglucose isomerase), RAG6 (KlPDC coding for pyruvate decarboxylase), and 18S transcripts are described in MATERIALS AND METHODS. As already known, two mRNA are detected for HGT1 (BILLARD et al. 1996). 18S mRNA was used as an internal standard.

 
The reduction of the transcription of genes encoding glycolytic enzymes and glucose transporters could result from a direct effect on these genes or it could be indirect, possibly through effects on the expression of genes encoding their regulators. One of these regulators—the glucose sensor, a positive regulator of RAG1—is encoded by RAG4 (BETINA et al. 2001). Another one, SCK1, codes for a helix-loop-helix type DNA-binding transcription factor, homologous to SGC1 of S. cerevisiae, which is required for full expression of glycolytic genes and of the glucose carrier gene RAG1 (LEMAIRE et al. 2002). KlGCR1 and KlGCR2 of K. lactis are the orthologs of the positive regulatory genes GCR1 and GCR2 of S. cerevisiae (HAW et al. 2001; NEIL et al. 2004). The KlGCR1 and KlGCR2 genes, like SCK1, appear to positively control the expression of glycolytic genes and RAG1 (NEIL et al. 2004). The amount of each of these transcripts was decreased compared to the wild-type strain in the enolase mutant strain (Figure 6). The transcript levels of these genes were reduced approximately five- to seven-fold in the mutant relative to the reference KlACT and KlAAC transcripts (data not shown).

View larger version (32K): In this window In a new window Download PPT slide   FIGURE 6.— Northern blot analysis of KlGCR1, KlGCR2, SCK1, and RAG4 transcription in Kleno null mutant. Approximately 5 µg of poly(A+) mRNA prepared from cells grown on 2% glucose was loaded in each slot. Strains used were MW270-7B (KlENO) and MWK3 (Kleno1::kanMX4). The probes used are described in MATERIALS AND METHODS.

 

A robust glycolytic flux is necessary for the full expression of RAG1:

KlENO is the second glycolytic gene controlling the RAG1 regulation to be identified. The RAG5 gene, coding for the single hexokinase in K. lactis, was already known to be required for the full expression of RAG1 (PRIOR et al. 1993). Enolase and hexokinase are suspected to harbor both catalytic and regulatory functions (BISSON and FRAENKEL 1983; ENTIAN and FRöHLICH 1984; PRIOR et al. 1993; FEO et al. 2000; SUBRAMANIAN and MILLER 2000), but our data suggest a more general hypothesis: the overall glycolytic flux may regulate glucose transport.

To investigate this hypothesis, we analyzed the expression of RAG1 in several mutants that are defective for different steps of the glycolytic or fermentation pathways (Figure 7A): Klhxk (hexokinase), Klpgi (phosphoglucoisomerase), Klpgk (phosphoglycerate kinase), Kleno (enolase), and Klpdc (pyruvate decarboxylase). The results showed that RAG1 expression is significantly reduced in Klhxk, Klpgk, and Kleno mutants (Figure 7B) in which the glycolytic flux is blocked. However, the Klpgi mutant, which can bypass the glycolytic block through the pentose phosphate pathway (JACOBY et al. 1993; GONZALEZ SISO et al. 1996), has little or no effect. The Klpdc mutation that blocks the first step of fermentation following glycolysis has no impact on RAG1 transcription. These findings suggest that glycolytic flux is required for full expression of RAG1.

View larger version (27K): In this window In a new window Download PPT slide   FIGURE 7.— Northern blot analysis of RAG1 in mutant affected at different steps of glycolysis (A). Approximately 5–10 µg of total RNA extracted from cells grown on 2% glucose was loaded in each slot. Electrophoresis conditions were as in Figure 3. The probes used to detect RAG1 and 18S transcripts are described in MATERIALS AND METHODS. (B) The strains MW270-7B, PM4-4B, and PM6-7A are the isogenic wild-type strains of the different mutants used. Klhxk, MWK11 strain; Klpgi, MWK12 strain; Klpgk, MWK43 strain; Kleno, MWK3 strain; Klpdc, MWK13 strain. These strains are described in Table 1. 18S mRNA was used as an internal standard. The hybridization signals have been quantified with a phosphoimager (MATERIALS AND METHODS) and numbers below the panel indicate the ratio of RAG1:18S.

 
Interestingly, regulation of glucose uptake by glycolytic flux in S. cerevisiae has been suggested (BISSON et al. 1993). We tested whether the activation of HXT1 (most closely related to RAG1) expression is impaired in the enolase mutant of S. cerevisiae grown on glucose. No effect on expression of an HXT1-LacZ fusion was observed in the single eno1 or eno2 mutants (data not shown). This negative result is not necessarily conclusive, because both single mutants retain some enolase activity (Figure 3A). Unfortunately, this experiment cannot be performed with the double-mutant eno1 eno2 since this mutant cannot grow on glucose (see Figure 2).

A reduced growth rate does not affect RAG1 transcription:

The glycolysis block in the Kleno mutant leads to a severe growth defect (Figure 2). The Klhxk and Klpgk mutants, but not the Klpgi and Klpdc mutants, show a similar growth defect (GOFFRINI et al. 1991; PRIOR et al. 1993; BIANCHI et al. 1996; data not shown). Thus, it remained possible that the RAG1 transcriptional defect in the Klhxk, Klpgk, and Kleno mutants could be due to their reduced growth rate rather than to their reduced glycolytic capability. To investigate this possibility, a wild-type strain (MW270-7B, KlENO) was grown in YPG with or without different growth inhibitors. We used antimycin A and potassium cyanide (KCN), which block the respiratory chain reaction, and geneticin (G418), which inhibits protein synthesis. Figure 8A shows that these compounds inhibit the growth of K. lactis on YPG. However, Northern blot analysis (Figure 8B) demonstrated that the RAG1 gene is still inducible by glucose in nondividing cells in the presence of antimycin A, KCN, or G418. This demonstrates that the RAG1 gene is induced to similar levels in dividing and nondividing cells. These results support the idea that the defect in RAG1 transcription in the Kleno, Klhxk, and Klpgk mutants is caused by reduced glycolytic flux rather than by impaired growth.

View larger version (31K): In this window In a new window Download PPT slide   FIGURE 8.— Effect of the growth rate on RAG1 transcription. (A) Inhibition of K. lactis growth by antimycin A, KCN, or G418. The MW270-7B (KlENO) strain was first grown in 50 ml YP medium containing 2% glycerol to an OD600 of 2. Cells were harvested, washed, and resuspended in sterile cold water and aliquots (15 OD600) were diluted in 50 ml YPG (2% glucose) or YPG containing 5 µM antimycin A, 5 mM KCN, or 100 µg/µl G418. Cultures were kept agitated at 28° during 10 hr and OD600 was checked at 0, 3, 6, and 10 hr. (B) Northern blot analysis of RAG1, KlACT, and 18S transcript levels. Total RNA was extracted from the YPG culture at t = 0 (shift on glucose) and from the four cultures at t = 10 hr. Electrophoresis conditions and quantification were done as described in the Figure 6 legend. The probes used to detect RAG1, KlACT, and 18S transcripts are described in MATERIALS AND METHODS. KlACT and 18S mRNA was used as internal standards and numbers below the corresponding panels indicate the ratio of RAG1:KlACT or RAG1:18S.

 

We have shown that the KlENO gene, which encodes enolase, the glycolytic enzyme that catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate, is required for normal regulation of expression of RAG1, encoding the low-affinity glucose permease in K. lactis. Glycolytic enzymes have been extensively studied and characterized at the structural and biochemical level. Recently, interest in glycolytic enzymes has been revived due to their implications in other biological pathways. In S. cerevisiae, the hexokinase PII, encoded by the HXK2 gene, is involved in glucose repression (RANDEZ-GIL et al. 1998). In fact, nuclear localization of Hxk2 appears to be glucose regulated, and it interacts in vivo with Mig1, the transcriptional repressor of many glucose-repressed genes (AHUATZI et al. 2004). In addition, in K. lactis and S. cerevisiae, hexokinase is also required for full glucose induction of RAG1 and HXT gene expression (PRIOR et al. 1993; ÖZCAN and JOHNSTON 1999). Several pieces of evidence suggest that enolase is a multifunctional protein playing a crucial role in transcriptional and physiological processes (reviewed in PANCHOLI 2001). For example, enolase has been identified as a heat-shock protein in S. cerevisiae (IIDA and YAHARA 1985) and has been found to have transcriptional regulatory functions in larger eukaryotes. In the latter case, these functions occur through binding of enolase to certain gene promoters: c-myc (FEO et al. 2000; SUBRAMANIAN and MILLER 2000) or STZ/ZAT10, encoding a transcriptional repressor in Arabidopsis (LEE et al. 2002).

The finding that the expression of RAG1 as well as several genes encoding glycolytic enzymes is affected in the Kleno mutant suggests that enolase could play a regulatory role in K. lactis in addition to its catalytic activity. However, we have not yet demonstrated a direct role for enolase in the transcriptional regulation of RAG1. In addition to enolase it was already known that induction of RAG1 expression by glucose is dependent on hexokinase activity (PRIOR et al. 1993). Since the loss of any one of the glycolytic steps tested, except that of phosphoglucoseisomerase, severely reduces RAG1 transcription, we believe that glucose metabolism generates a signal that induces RAG1 expression. We cannot, however, exclude the possibility that enolase has a general regulatory function on other genes.

The defect in RAG1 transcription in mutants blocked in glycolysis suggests the existence of regulatory mechanisms that prevent expression of genes encoding glucose transporters if a functional glycolytic pathway cannot be maintained. MILKOWSKI et al. (2001) previously showed that the absence of glucose transporters (hence glucose uptake) impaired the induction of KHT1/RAG1 expression by high levels of glucose. Altogether, these data suggest that an intracellular glucose-sensing mechanism relying on glucose metabolism through glycolysis may ensure optimal glucose uptake by activating expression of the gene encoding the low-affinity glucose transporter. This intracellular pathway presumably collaborates with the extracellular glucose-sensing mechanism operating through the Rag4 glucose sensor in the cell membrane (BETINA et al. 2001). In E. coli, the expression of ptsG, encoding the major glucose transporter IICB^Glc, also requires glycolytic flux (KIMATA et al. 2001). Whatever the mechanism, regulation of glucose uptake by glycolytic flux seems to have been conserved from bacteria to yeasts.

We have a few clues to the mechanism by which glycolytic flux regulates expression of RAG1 and glycolytic genes. The inactivation of KlENO leads to a severe reduction in expression of the regulatory genes KlGCR1, KlGCR2, SCK1, and RAG4 (Figure 6). Thus, the effects of enolase on expression of genes encoding glycolytic enzymes and glucose transporters may be indirect. KlGCR1, KlGCR2, and SCK1 are required for the full expression of glycolytic genes (LEMAIRE et al. 2002; NEIL et al. 2004). The KlGcr1/KlGcr2 complex directly regulates glycolytic gene expression through binding of KlGcr1 to glycolytic gene promoters (NEIL et al. 2004). An interaction between Sck1 and K. lactis glycolytic promoters is also probable since its S. cerevisiae ortholog (Sgc1) binds to the ENO1 promoter (SATO et al. 1999). Hence, the transcriptional defect of RAG5/RAG6 genes in the Kleno mutant is almost certainly the consequence of the low level of expression of KlGCR1, KlGCR2, and SCK1. Interestingly, these transcription factors are also required for the full glucose induction of RAG1 expression (LEMAIRE et al. 2002; NEIL et al. 2004). At least in the case of KlGcr1/KlGcr2, this effect seems indirect since KlGcr1 does not bind the RAG1 promoter in vitro (NEIL et al. 2004) and thus may be a consequence of the reduced glycolytic flux in the Klgcr1/Klgcr2 mutants. Although we cannot exclude a cumulative effect of the reduced expression of KlGCR1, KlGCR2, SCK1, and RAG4 on RAG1 expression, we favor the hypothesis that a product of glycolysis may control the activity of an unidentified regulator. Such metabolic controls have been described already in S. cerevisiae. For instance, the Mcm1 transcriptional regulator is regulated post-transcriptionally by the glycolytic flux (CHEN and TYE 1995). Moreover, the efficient transcription of yeast AMP biosynthetic genes requires interaction between the transcription factors Bas1p and Bas2p, and this interaction is promoted in the presence of a metabolic intermediate (SAICAR) of this biosynthetic process (REBORA et al. 2001).

On the basis of the present findings, we propose that the regulation of RAG1 expression by glucose involves two pathways: (i) a pathway involving the glucose sensor Rag4 that responds to extracellular glucose availability (BETINA et al. 2001) and (ii) a pathway responding to an intracellular signal generated by glycolysis. Together, these pathways can be considered to be an autoregulatory device for the fermentative utilization of sugars in yeast. The K. lactis system with its nonredundancy of the genes of glucose metabolism, the clear Rag^– phenotype associated with glycolytic mutations, and its metabolic properties appears to be a suitable tool to study intracellular glucose sensing in yeast.

We thank Melanie Annat for cloning of the KlENO gene and Hiroshi Fukuhara for critical reading of the manuscript.

Sequence data from this article have been deposited with the EMBL Data Library under accession no. AJ586240.

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