ATP SYNTHASE OF YEAST MITOCHONDRIA - ISOLATION AND DISRUPTION OF THE ATP-EPSILON-GENE

The nuclear gene encoding the subunit epsilon of the catalytic sector F1 of the yeast Saccharomyces cerevisiae ATP synthase was cloned and sequenced. Degenerated oligonucleotide primers were constructed from primary structure data. A part of the ATPepsilon gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence a nondegenerated oligonucleotide probe was constructed and used for isolating a 2040-base pair EcoRI fragment bearing the whole gene. A 186-base pair open reading frame encoding a 62-amino acid polypeptide is described. The deduced amino acid sequence was one amino acid longer than the mature protein. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity. The catalytic sector appeared unstable during purification but F0-subunits were still bound to F1. The mutation promoted a highly oligomycin-sensitive uncoupling of the mitochondrial respiration rate.