We introduced mutations to test the function of the hydrophobic sector of subunit 4 from Saccharomyces cerevisiae ATP synthase. Mutations were introduced at the chromosomic locus by homologous transformation of a strain disrupted in the ATP4 gene. The strain carrying the replacement Leu68-Val69-->Arg-Glu did not grow at 37 degrees C owing to a lack of assembly of F1 and Fo sectors at this temperature. The mutant strain grew slowly by oxidative phosphorylation at 28 degrees C with a growth yield 30% lower than the wild type. Analysis of the mutant strain showed a homogeneous population of altered ATP synthase with an energy coupling impairment. The mutant strain was oligomycin-resistant since the I50 value of oligomycin inhibition of ATPase and ATP synthase activities was 2-3-fold higher than that of the wild type, thus showing an alteration of the target to oligomycin. The level of phosphorylation or ATP induced a proton-dissipating pathway through Fo, which was insensitive to oligomycin but was sensitive to dicyclohexylcarbodiimide, thus suggesting an alteration in the regulation of ATP synthase proton permeability by the catalytic sector. From these results, we propose that the dicyclohexylcarbodiimide inhibition site is located upstream of the oligomycin inhibition site when considering the proton flux occurring during ATP synthesis.