The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.