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 ATP epsilon 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.
Guélin
E
E
Institut de Biochimie Cellulaire, Université de Bordeaux II, France.
Chevallier
J
J
Rigoulet
M
M
Guérin
B
B
Velours
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology; D005800:Genes, Fungal; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D009840:Oligomycins / Q000494:pharmacology; D015345:Oligonucleotide Probes; D010101:Oxygen Consumption / Q000187:drug effects; D010957:Plasmids; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D016385:TATA Box
D004271:DNA, Fungal; D046911:Macromolecular Substances; D009840:Oligomycins; D015345:Oligonucleotide Probes; D006180:Proton-Translocating ATPases