MUTATION IN THE HYDROPHOBIC DOMAIN OF ATP SYNTHASE SUBUNIT-4 (SUBUNIT-B) OF YEAST MITOCHONDRIA DISTURBS COUPLING BETWEEN PROTON TRANSLOCATION AND CATALYSIS

We introduced mutations to test the function of the hydrophobic sector of subunit 4 from Saccharomyces cerevisiae ATP synthase. Mutations we re introduced at the chromosomic locus by homologous transformation of a strain disrupted in the ATP4 gene. The strain carrying the replacem ent Leu68-Val69--> Arg-Glu did not grow at 37 degrees C owing to a lac k of assembly of F-1 and F-o sectors at this temperature. The mutant s train grew slowly by oxidative phosphorylation at 28 degrees C with a growth yield 30% lower than the wild type. Analysis of the mutant stra in showed a homogeneous population of altered ATP synthase with an ene rgy coupling impairment. The mutant strain was oligomycin-resistant si nce the I-50 value of oligomycin inhibition of ATPase and ATP synthase activities was 2-3-fold higher than that of the wild type, thus showi ng an alteration of the target to oligomycin. The level of phosphoryla tion or ATP induced a proton-dissipating pathway through F-o, which wa s insensitive to oligomycin but was sensitive to dicyclohexylcarbodiim ide, thus suggesting an alteration in the regulation of ATP synthase p roton permeability by the catalytic sector. From these results, we pro pose that the dicyclohexylcarbodiimide inhibition site is located upst ream of the oligomycin inhibition site when considering the proton flu x occurring during ATP synthesis.