Impact of mitochondrial function on yeast susceptibility to antifungal compounds

Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.     

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.     

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.     

sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.     

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.     

Transformation of Kluyveromyces fragilis

For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K. fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously. The same plasmid could also be used to transform S. cerevisiae trp1 mutant to Trp+. Thus, KARS2 of K. lactis enables the vector to replicate in K. fragilis, K. lactis, and S. cerevisiae, whereas ARS1 of S. cerevisiae allows autonomous replication only in S. cerevisiae.     

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Maintenance of mitochondrial morphology is linked to maintenance of the mitochondrial genome in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.     

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.     

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.     

The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.     

Impact of mitochondrial activity on the cell wall composition and on the resistance to tannic acid in Saccharomyces cerevisiae

In screening for resistance to tannic acid, mutants of Saccharomyces cerevisiae with an altered cell wall composition were recently isolated. Here we show that these mutants were all respiratory deficient. Cytoplasmic petite mutants isolated after ethidium bromide mutagenesis were resistant to tannic acid and had cell wall characteristics similar to the mutants isolated by screening for tannic acid resistance as shown by the lower sensitivity to zymolyase, a cell wall hydrolyzing enzyme, and by a changed sensitivity to calcofluor white, a molecule interfering with the cell wall assembly. Reintroducing active mitochondria to a tannic-acid-resistant mutant reduced the tannic acid resistance and zymolyase resistance to the wild-type level, showing that a mitochondrial mutation was responsible for the changes in cell wall composition and in tannic acid sensitivity.