Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase

By means of a yeast genome database search, we have identified an open read ing frame located on chromosome XVI of Saccharomyces cerevisiae that encode s a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bov ine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of t he HIS3 gene into the coding region of ATP20, retained oxidative phosphoryl ation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cell s expressing a variant of subunit g with a hexahistidine motif indicated th at this polypeptide was associated with other well-characterized subunits o f the yeast complex. Whilst mitochondria isolated from the atp20-null strai n had the same oxidative phosphorylation efficiency (ATP : O) as that of th e control strain, the atp20-null strain displayed approximately a 30% reduc tion in both respiratory capacity and ATP synthetic rate. The absence of su bunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating AT P synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of re spiration, ATP synthesis and cytochrome c oxidase activity in yeast.