Single Copies of Subunits d, Oligomycin-sensitivity Conferring Protein, and b Are Present in the Saccharomyces cerevisiaeMitochondrial ATP Synthase

Abstract

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex 6 -tagged subunit can be isolated via Ni 2؉ -nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14 -18). Strains were constructed in which Hex 6 -tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex 6 -tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni 2؉ -nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex 6 -tagged subunit. As only the Hex 6 -tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.

Comparative Genomics of Rickettsia prowazekii Madrid E and Breinl Strains

“…CYCJ might be required for the biogenesis of c-type cytochromes in Bradyrhizobium japonicum and Rhizobium etli (33,41). In Drosophila melanogaster and Saccharomyces cerevisiae, OSCP was implicated in proton conduction during ATP synthesis (4,9,34). Cytochrome c is believed to be an important factor in the initiation of the apoptotic cascade.…”

Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae

“…Bands containing mtATPase complexes were excised and subjected to second dimension SDS-PAGE, and gels silver stained (C). Subunit assignments were based on their mobility within the gel relative to size standards and by comparison with published mtATPase subunit profiles (Bateson et al, 1999). Lanes correspond to the numbered lanes from A, with lower case letters referring to mtATPase species a-d (above).…”

“…Polyclonal antibodies against subunits e and g were kindly provided by Jean Velours (Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Segalen, Bordeaux, France). Membranes were incubated with alkaline-phosphate conjugated antibodies (AmradPharmacia, Melbourne, Australia) and visualised using a Storm Phosphoimager (Molecular Dynamics Australia, Melbourne) following the addition of chemifluorescent Vistra substrate (Amersham Pharmacia Biotech, Sydney, Australia) as described previously (Bateson et al, 1999).…”

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

“…In the structurally less complex ATP synthase of Escherichia coli the stator stalk is composed of subunit ␦ and two copies of subunit b (17). However, only a single copy of subunit b is present in the mtATPase of the yeast Saccharomyces cerevisiae (18); thus, in conjunction with OSCP (the mitochondrial homolog of subunit ␦), other F 0 subunits contribute to form the stator stalk. Second, movement of protons through the membrane-embedded channel of F 0 allows conversion of the proton gradient generated by respiratory chain activity into chemical energy, in the form of ATP made by phosphorylation of ADP on F 1 .…”