RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1 . Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C₁₈ fatty acid. The KlLAC1 protein preferred long-chain (C₁₈) fatty acids to very-long-chain (C₂₆) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.     

Suppression of a nuclear frameshift mutation by a mitochondrial tRNA in the yeast Kluyveromyces lactis

A fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine-tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory-deficient mutant of this yeast. The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al. 2000 Yeast 16: 307-314). We report here that the mitochondrial tRNAVAL gene, when transformed into K. lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA. In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt-tRNAVAL from the nucleus. Suppression could occur through a non-canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation. This is a new case of informational suppression in that the suppression of a chromosomal mutation is not caused by a second mutation but to a mislocalization/expression of a mt-tRNA.     

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.