Mutational bisection of the mitochondrial DNA stability and amino acid biosynthetic functions of ilv5p of budding yeast.
Ilv5p is a bifunctional yeast mitochondrial enzyme required for branched chain amino acid biosynthesis and for the stability of mitochondrial DNA (mtDNA) and its parsing into nucleoids. The latter occurs when the general amino acid control (GAC) pathway is activated. We have isolated ilv5 mutants that lack either the enzymatic (a(-)D(+)) or the mtDNA stability function (a(+)D(-)) of the protein. The affected residues in these two mutant classes cluster differently when mapped to the 3-D structure of the spinach ortholog of Ilv5p. a(-)D(+) mutations map to conserved internal domains known to be important for substrate and cofactor binding, whereas the a(+)D(-) mutations map to a C-terminal region on the surface of the protein. The a(+)D(-) mutants also have a temperature-sensitive phenotype when grown on a glycerol medium, which correlates with their degree of mtDNA instability. Analysis of an a(+)D(-) mutant with a strong mtDNA instability phenotype shows that it is also unable to parse mtDNA into nucleoids when activated by the GAC pathway. Finally, the wild-type Escherichia coli ortholog of Ilv5p behaves like a(+)D(-) mutants when expressed and targeted to mitochondria in ilv5Delta yeast cells, suggesting that yeast Ilv5p acquired its mtDNA function after the endosymbiotic event.
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
Joseph M
JM
Bateman
Philip S
PS
Perlman
Ronald A
RA
Butow
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005816:Genetic Complementation Test; D005838:Genotype; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D017931:DNA Primers; D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D000429:Alcohol Oxidoreductases; C470636:Ilv5 protein, S cerevisiae