ATP SYNTHASE OF YEAST MITOCHONDRIA - ISOLATION OF THE F1 DELTA-SUBUNIT, SEQUENCE AND DISRUPTION OF THE STRUCTURAL GENE

The delta-subunit was isolated from the purified yeast F-1. Partial pr otein sequences were determined by direct methods. From this informati on, degenerated primers were constructed. A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA. Fr om the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6-kbp BamHI-EcoRI DNA fragment bearing the whole gene. A 1036-bp DraI fragment was sequenced. A 480-bp open r eading frame encoding a 160-amino-acid polypeptide is described. The d educed amino acid sequence is 22 amino acids longer than the mature pr otein, which is 138 amino acids long with a mass of 14555 Da. The delt a-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 6 6%, 61% and 92% similar to the epsilon-subunit of Escherichia coli and the delta-subunits of beef heart and Neurospora crassa, respectively. A null mutant was constructed. The mutation was recessive and dramati cally affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho(-), ATP delta::U RA3) displayed low or no ATPase activity with an unstable catalytic se ctor, since a polyclonal antibody directed against the beta subunit di d not coprecipitate the alpha subunit.