The C-terminal region of subunit 4 (subunit b) is essential for assembly of the F0 portion of yeast mitochondrial ATP synthase.
The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 degrees C this strain showed the same phenotype as above. When grown at permissive temperature (30 degrees C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30 degrees C and 37 degrees C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.
Paul
M F
MF
Institut de Biochimie Cellulaire du Centre National de la Recherche Scientifique, Université de Bordeaux II, France.
Guerin
B
B
Velours
J
J
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D001483:Base Sequence; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009840:Oligomycins / Q000494:pharmacology; D009841:Oligonucleotides / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D011233:Precipitin Tests; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development
D003470:Culture Media; D009840:Oligomycins; D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D010100:Oxygen