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

Abstract

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...

The Subunit F of Mitochondrial Yeast ATP Synthase

“…ATPase activities of isolated mitochondria. Mitochondria were isolated from cells grown at 28°C with 2% lactate or glycerol as carbon source for the wild-type and complemented SVY15-1 cells and 2% galactose as carbon source for the null mutant SVY14 and the rhocells (the rho-mitochondria are presented as a control; Paul et al, 1989). Measurements were performed in triplicate.…”

“…This low respiration rate was mainly the consequence of a high rho-cell content. However, it was previously observed that the absence of a functional F, had an indirect effect on the assembly and activity of the cytochrome oxidase (Choo et al, 1985 ;Paul et al, 1989). Similarly, cytochrome oxidase activities of 0.05 pmol 0, .…”

“…Extraction and isolation of subunits 4, 6, 8 and 9 from whole mitochondria have been described previously (Paul et al, 1989). Protein concentrations were determined as in Lowry et al (1951) in the presence of SDS using bovine serum albumin as a standard.…”

The Saccharomyces cerevisiae ATP22 Gene Codes for the Mitochondrial ATPase Subunit 6-Specific Translation Factor

“…The slow growth of N417 on rich glycerol/ethanol medium suggests that the genetic lesion does not abolish expression of F 0 totally, which would explain why this strain is able to synthesize subunit 6. Like other ATPase mutants that display a pleiotropic deficiency of cytochrome oxidase (Marzuki et al 1989;Paul et al 1989), most of the atp22 mutants showed a substantial decrease in translation of subunit 1 of this respiratory complex.…”

“…Western analysis of ATPase subunits indicated that atp22 point mutants are grossly deficient in subunit 6 (Helfenbein et al 2003). A subunit 6 deficit is not necessarily related to translation of the protein or absence of the mRNA (Ellis et al 2004) but can also be caused by its turnover in mutants defective in F 0 assembly (Paul et al 1989(Paul et al , 2000Arselin et al 1996). In our earlier studies of the atp22 mutant, N417, some incorporation of 35 S-methionine into subunit 6 was observed when cells were labeled in vivo in the presence of cycloheximide to arrest cytoplasmic protein synthesis (Helfenbein et al 2003).…”