Specific mutations in alpha- and gamma-subunits of F1-ATPase affect mitochondrial genome integrity in the petite-negative yeast Kluyveromyces lactis.

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

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Mutations in MGI genes convert Kluyveromyces lactis into a petite-positive yeast.

Genetics ◽

10.1093/genetics/133.3.517 ◽

1993 ◽

Vol 133 (3) ◽

pp. 517-525 ◽

Cited By ~ 3

Author(s):

X J Chen ◽

G D Clark-Walker

Keyword(s):

Cytochrome C ◽

Mitochondrial Genome ◽

Ethidium Bromide ◽

High Frequency ◽

Kluyveromyces Lactis ◽

Genome Integrity ◽

Gene Products ◽

Targeted Disruption ◽

Nuclear Mutation ◽

Petite Negative Yeast

Abstract Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.

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Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

Genetics ◽

10.1093/genetics/154.1.147 ◽

2000 ◽

Vol 154 (1) ◽

pp. 147-154 ◽

Cited By ~ 1

Author(s):

Douglas J Kominsky ◽

Peter E Thorsness

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Schizosaccharomyces Pombe ◽

Atp Synthase ◽

Kluyveromyces Lactis ◽

Blot Hybridization ◽

Inner Mitochondrial Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Mitochondrial Atp Synthase ◽

Petite Negative Yeast

Abstract Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the α- and γ-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

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