Abstract
The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase.
Highlights
The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis
The experiments described in this paper focus on the reassessment of the polypeptide composition of the yeast ATP synthase using a different isolation technique, namely blue native electrophoresis (BN-PAGE)
BN-PAGE was repeated on the preparative scale using the hydroxyapatite fraction with the highest amount of ATP synthase complex (Fig. 1B, lane 4)
Summary
Vol 274, No 1, Issue of January 1, pp. 36 –40, 1999 Printed in U.S.A. (Received for publication, August 17, 1998, and in revised form, September 18, 1998). All components of the bovine catalytic sector of the ATP synthase (F1), i.e. subunits ␣, , ␥, ␦, ⑀, and the inhibitor protein (IF1), have homologous counterparts in Saccharomyces cerevisiae (4 –9). Known as Tim, was originally reported as being a component of the mitochondrial inner membrane import machinery [18]; it has subsequently been shown to be a membrane-bound subunit of the ATP synthase complex [17]. Combined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension, an overview on the protein subunits of all oxidative phosphorylation complexes is obtained in a two-dimensional gel [22] Using this two-dimensional electrophoretic technique, we observed in the ATP synthase the presence of a previously undetected protein in the 6 –7-kDa range. Su j is encoded by a gene termed here ATP18 and has no apparent bovine counterpart
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