The Rag4 glucose sensor is involved in the hypoxic induction of KlPDC1 gene expression in the yeast Kluyveromyces lactis.

Kluyveromyces lactis is a yeast which cannot grow under strict anaerobiosis. To date, no factors responsible for oxygen sensing and oxygen-dependent regulation of metabolism have been identified. In this paper we present the identification of the glucose sensor Rag4 as a factor essential for oxygen-dependent regulation of the fermentative pathway.

The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis.

The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.

Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p. Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p. Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9. HXT11 and HXT9, encoding nearly identical proteins, have a high degree of identity to monosaccharide transporters of the major facilitator superfamily (MFS). In this study, we show that the HXT11 product, which allows glucose uptake in a glucose permease mutant (rag1) strain of Kluyveromyces lactis, is also involved in the pleiotropic drug resistance process. Loss of HXT11 and/or HXT9 confers cycloheximide, sulfomethuron methyl, and 4-NQO (4-nitroquinoline-N-oxide) resistance. Conversely, HXT11 overexpression increases sensitivity to these drugs in the wild-type strain, an effect which is more pronounced in a strain having both PDR1 and PDR3 deleted. These data show that the two putative hexose transporters Hxt11p and Hxt9p are transcriptionally regulated by the transcription factors Pdr1p and Pdr3p, which are known to regulate the production of ABC transporters required for drug resistance in yeast. We thus demonstrate the existence of genetic interactions between genes coding for two classes of transporters (ABC and MFS) to control the multidrug resistance process.

Expression of human ANT2 gene in highly proliferative cells: GRBOX, a new transcriptional element, is involved in the regulation of glycolytic ATP import into mitochondria.

The adenine nucleotide translocator (ANT) is the most abundant mitochondrial inner membrane protein which catalyses the exchange of ADP and ATP between cytosol and mitochondria. The human ANT protein has three isoforms encoded by three differentially regulated nuclear genes. The ANT gene expression was examined in several human cells. The gene encoding the ANT2 isoform was found specifically induced in Simian virus 40 (SV40)-transformed, tumoral and mtDNA lacking rho degrees cell lines. Moreover, the ANT2 gene was preferentially expressed under a glycolytic metabolism. Functional complementation of a Saccharomyces cerevisiae mutant revealed that the human ANT2 protein specifically restores yeast cell growth under anaerobic conditions. Sequence analysis of the ANT2 proximal promoter in comparison to that of the third yeast adenine nucleotide translocator (AAC3) led us to identify a new motif termed GRBOX. Promoter-deletion transfection and mobility gel-shift assays revealed that this motif is recognized by a negative transcriptional regulator. This transcription factor might be involved in a molecular mechanism which selects the import of the glycolytic ATP in the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which depend strongly on glycolysis for ATP synthesis.

RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.

The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished.

Creation of a functional promoter by rearrangement in a Kluyveromyces lactis linear plasmid.

The expression of genes from cytoplasmic killer plasmids in the yeast Kluyveromyces lactis depends on their own specific transcription system. Therefore, the kanamycin/G418-resistance-encoding gene, KmR, under its natural promoter cannot be expressed when integrated into the pGKL1 plasmid. However, one G418R transformant clone was isolated. The resistance was due to the presence of two modified plasmids, k1-kan2a (10.4 kb) and k1-kan2b (5.2 kb) which were derivatives of pGKL1 containing the KmR gene. In these mutant plasmids, a large part of pGKL1 has been replaced by the KmR gene harboring a rearranged 5'-flanking region extending over 600 bp. This new DNA sequence has been cloned and sequenced. The rearranged sequence allowed the KmR gene to be expressed at high level, enabling the transformant cells to grow on a medium containing G418 at 2 mg/ml. This high level of resistance was found to be due to increased transcription of the KmR gene. Primer extension experiments suggested that the rearranged upstream region of KmR contained transcription promoting sites recognized by the killer-plasmid-specific transcription system.

The respiratory system of Kluyveromyces lactis escapes from HAP2 control.

A functional homolog of the Saccharomyces cerevisiae HAP2 gene, coding for one element of a transcriptional activator complex, was cloned from the yeast Kluyveromyces lactis and its nucleotide sequence was determined. Inactivation of the gene had no significant effect on respiration-dependent growth, suggesting that the HAP2/3/4 complex has no major control over the formation of the mitochondrial respiratory system in K. lactis.

Expression of a foreign KmR gene in linear killer DNA plasmids in yeast.

The killer plasmids of the yeast Kluyveromyces lactis, pGKL1 and 2 (k1 and k2 for short), are linear double-stranded DNAs. The expression of genes of these plasmids is thought to depend on their own transcription system. Cloning the plasmid genes in conventional circular vectors is therefore not suitable for transcriptional studies, because such vectors use the host nuclear transcription system. In vitro modification of the linear plasmid genomes in order to introduce transcription reporter genes has been difficult because the structure of the plasmids, with covalently bound terminal proteins, does not allow their manipulation in vitro and amplification in Escherichia coli. We introduced the kanamycin/G418 resistance gene, KmR, into the k1 plasmid in vivo, by transforming the yeast with the linearized KmR gene bordered with short k1 sequences (part of the region encoding the toxin) to allow homologous recombination with the resident k1. In the linear recombinants obtained, however, the KmR was not expressed, while it was expressed if carried on circularized plasmids. By replacing the native promoter of KmR by the ORF1 promoter from k1, the KmR gene could be expressed in linear recombinants and conferred on the host a high level of resistance to the drug. All the linear recombinant plasmids were extremely stable under nonselective conditions. As a rare event, the integration of KmR produced a palindromic rearrangement of the k1 plasmid.

The Kluyveromyces lactis KEX1 gene encodes a subtilisin-type serine proteinase.

KEX1 is a chromosomal gene required for the production of the killer toxin encoded by the linear DNA plasmid pGKL-1 of Kluyveromyces lactis. The nucleotide sequence of the cloned KEX1 gene has been determined. The deduced structure of the KEX1 protein, 700 amino acids long, indicated that it contained an internal domain with a striking homology to the sequences of the subtilisin-type proteinases, and a probable transmembrane domain near the carboxyl terminus. The results confirm the hypothesis that the product of the gene KEX1 of K. lactis is a proteinase involved in the processing of the toxin precursor.

Genomic exploration of the hemiascomycetous yeasts: 11. Kluyveromyces lactis.

Random sequencing of the Kluyveromyces lactis genome allowed the identification of 2235-2601 open reading frames (ORFs) homologous to S. cerevisiae ORFs, 51 ORFs which were homologous to genes from other species, 64 tRNAs, the complete rDNA repeat, and a few Ty1- and Ty2-like sequences. In addition, the complete sequence of plasmid pKD1 and a large coverage of the mitochondrial genome were obtained. The global distribution into general functional categories found in Saccharomyces cerevisiae and as defined by MIPS is well conserved in K. lactis. However, detailed examination of certain subcategories revealed a small excess of genes involved in amino acid metabolism in K. lactis. The sequences are deposited at EMBL under the accession numbers AL424881-AL430960.

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.

Genomic exploration of the hemiascomycetous yeasts: 19. Ascomycetes-specific genes.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific'. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes. Half of the 'Ascomycetes-specific' genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

Genomic exploration of the hemiascomycetous yeasts: 21. Comparative functional classification of genes.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.

The RAG3 gene of Kluyveromyces lactis is involved in the transcriptional regulation of genes coding for enzymes implicated in pyruvate utilization and genes of the biosynthesis of thiamine pyrophosphate.

The RAG3 gene of Kluyveromyces lactis, a homolog of PDC2 of Saccharomyces cerevisiae, is known to be a regulator of the pyruvate decarboxylase gene KlPDC1. We have identified new target genes for Rag3p. The RAG3 gene product was found to be required for the transcription of two genes of the biosynthetic pathway of thiamine (a cofactor of pyruvate decarboxylase). Conversely, the RAG3 gene product partially repressed the expression of the pyruvate dehydrogenase gene KlPDA1. Therefore, RAG3 may act as a general regulator in the balance of the two alternative pathways of pyruvate metabolism in yeast.

A palindromic mutation of the linear killer plasmid k2 of yeast.

Production of the killer toxin in Kluyveromyces lactis is dependent on the presence of two linear DNA plasmids, k1 and k2. We isolated a non-killer mutant, VM5, with a modified plasmid composition. It had lost k1, but conserved k2, and acquired, in addition, three new DNA species. The new species were found to be rearranged derivatives of the k2 plasmid. One of them, pVM5-1, was made of the left terminal 4720 bp sequence of k2, including the inverted terminal repeat, and was organized as a large palindromic dimer molecule. The second, pVM5-2, was made of one strand of the pVM5-1 palindrome, folded into a hairpin structure. Like normal k2, pVM5-1 and 2 were present in a high copy number. The third species, pVM5-x, of variable size, was also a deletion product of k2, but not palindromic, and did not contain the terminal repeat. Genetic analysis showed that the presence of the palindromic derivatives appeared to destabilize the normal k2 genome, leading to gradual accumulation of plasmid-less cells.