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 F1. Partial protein sequences were determined by direct methods. From this information, degenerated primers were constructed. A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA. From 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 reading frame encoding a 160-amino-acid polypeptide is described. The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14,555 Da. The delta-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 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 dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho-, ATP delta::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the beta subunit did not coprecipitate the alpha subunit.
Institut de Biochimie et Génétique Cellulaire, Centre National de la Recherche Scientifique, Université de Bordeaux II, France.
M F
MF
Giraud
J
J
Velours
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005784:Gene Amplification; D005800:Genes, Fungal; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet; D016385:TATA Box
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D006180:Proton-Translocating ATPases