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 D273-10R/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 oligomycininsensitive and the F, sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of Fo revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of Fo. 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.ATP synthase is composed of subunits encoded by two genetic systems. In Saccharom,vces cerevisiae, the soluble portion of the enzyme, F1, contains the catalytic site for synthesis and hydrolysis of ATP. It is composed of five subunits (a, p, y, 6, E ) [l] that are encoded by nuclear DNA translated in the cytoplasm [2] and imported into the organelles [3]. The membranous portion of the complex, named Fo, catalyses H + conduction across the membrane and confers oligomycinsensitivity to F1 [4]. This membranous domain contains three highly hydrophobic mitochondrial gene products (subunits 6, 8 and 9) [5-81. Three additional nuclear-encoded polypeptides participate in the complex [l, 9-111 and presumably constitute a link between F1 and the three highly hydrophobic polypeptides of Fo. Electrophoresis shows their apparent molecular masses to be 25, 21 and 19 kDa respectively. In yeast, the structure of subunit 4 (25 kDa) [12] and the oligomycinsensitivity-conferring protein (21 kDa) [13] were recently reported.In previous papers, we have reported the purification procedure for subunit 4 and the sequence of the nuclear gene ATP4 [12, 141. Mature subunit 4 contains 209 amino acid residues/molecule and the predicted molecular mass is 23250 Da. A high similarity between subunit 4 and the b subunits of E. cofi and beef heart mitochondrial ATP synthases was reported [15].As revealed by secondary structure prediction methods, two distinct domains should compose subunit 4. The hydrophobic N-terminal part and the hydrophilic C-terminal sector Enzyme. ATP synthase or ATP phosphohydrolase (EC 3.6.1.34).confer amphiphilic behaviour to this subunit. Cross-linking experiments were previously reported between subunit 4 and F1 subunits c1 and b, and Fo subunits 9 and 6 [16, 171. Proteolytic cleavage of bovine submitochondrial particles depleted in F...
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