Selection of the nuclear gene for the mitochondrial adenine nucleotide translocator by genetic complementation of the op1 mutation in yeast.

Abstract

Oxidative phosphorylation 1 (OP1), a nuclear gene of yeast Saccharomyces cerevisiae which is required for the expression of a functional mitochondria, has been isolated on a recombinant plasmid. The gene was selected from a recombinant plasmid pool which contained wild type yeast genomic DNA by transformation of the yeast nuclear mutant (op1) followed by a two-stage screening procedure. A recombinant plasmid containing a 2.6 kilobase Bam HI fragment of genomic yeast DNA inserted in either orientation into the single Bam HI site of yeast vector YEp 13 could complement the op1 mutation. Analysis of the gene product of this inserted DNA by three independent methods, 1) in vivo expression in Escherichia coli maxicells, 2) cell-free translation of plasmid selected RNA, and 3) expression analysis in yeast, revealed that its gene product is a protein of Mr = 30,000-32,000, which cross-reacts with specific anti-serum to the adenine nucleotide translocator of the mitochondrial inner membrane. The selection procedure is efficient and can be used for the isolation of any defined yeast nuclear gene which participates in mitochondrial development.