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A vital function for mitochondrial DNA in the petite-negative yeast Kluyveromyces lactis
1Molecular and Population Genetics Group, Research School of Biological Sciences, Australian National University, Canberra City, Australia.
Abstract:
Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MG12 and MG15 encoding the alpha- and gamma-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost. In this study we show that wild-type K. lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned the K. lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase. These observations suggest that mtDNA in K. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of Fo.
Insights
Petite-negative yeasts like Kluyveromyces lactis require mitochondrial DNA (mtDNA) for survival. Specific mutations allow mtDNA loss by suppressing lethality, revealing mtDNA
Area of Science:
- Molecular and Cellular Biology
- Yeast Genetics
- Mitochondrial Biology
Background:
- Petite-negative yeasts are resistant to mitochondrial DNA (mtDNA) loss induced by DNA-targeting drugs.
- Specific mutations in Kluyveromyces lactis nuclear genes MG12 and MG15 permit mtDNA loss.
- These genes encode subunits of the mitochondrial F1-ATPase, an essential enzyme complex.
Purpose of the Study:
- To investigate the role of mitochondrial DNA (mtDNA) in the survival of the petite-negative yeast Kluyveromyces lactis.
- To elucidate the function of mgi mutations in suppressing lethality associated with mtDNA loss.
- To identify the vital function encoded by mtDNA in K. lactis.
Main Methods:
- Assessed the viability of wild-type K. lactis strains with and without functional mtDNA.
- Examined the tolerance of mgi mutant alleles in the absence of mtDNA using multicopy plasmids.
- Cloned and disrupted the K. lactis MGM101 homologue in diploid and haploid strains.
- Analyzed segregants for growth defects and cell viability following gene disruption.
Main Results:
- Wild-type K. lactis cannot survive without its mitochondrial genome.
- mgi mutations suppress the lethality resulting from mtDNA loss.
- Disruption of the MGM101 homologue leads to inviable cells in wild-type backgrounds but is tolerated in mgi mutant strains.
Conclusions:
- mtDNA encodes a vital function essential for K. lactis survival.
- mgi mutations confer tolerance to mtDNA loss by overcoming this essentiality.
- The vital mtDNA-encoded function may reside in the mitochondrially encoded subunits of the Fo complex.