RELOCATION OF URF A FROM THE MITOCHONDRION TO THE NUCLEUS CURES THE MITOCHONDRIAL MUTATOR PHENOTYPE IN THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE

In previous papers we have reported the characterisation of mitochondr ial mutator mutants of Schizosaccharomyces pombe. In contrast to nucle ar mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformat ion sys tem for fission yeast mitochondria is not yet available, we re located the mitochondrial urf a gene to the nucleus. As host strain fo r the ectopic expression, we used the nonsense mutant ana'-6, which ca rries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving ri se to fully respiration competent colonies, colonies that show moderat e growth on glycerol and a fraction of colonies that are unable to gro w on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic e xpression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types o f expression cassettes containing either the mitochondrial version of the urf a gene (mf-urf a) or a standard genetic code version (nc-urf a ; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the express ion of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana'-6, whereas the expression of th e nc-urf a gene completely restores the wild-type (non-mutator) phenot ype. The significant similarity of the urf a gene to the mitochondrial var 1 gene of S. cerevisiae and homologous genes in other yeasts sugg ests that the urf a gene product might be a ribosomal protein with a d ual function in protein synthesis and maintenance of mitochondrial DNA integrity.