Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants, thiol modification and cross-linking reagents.
A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits. The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents. Our data suggest interactions between the first transmembranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes.
Velours
J
J
Institut de Biochimie et Génétique Cellulaires du CNRS, 1 rue Camille Saint Saëns, 33077, cedex, Bordeaux, France. john.velours@ibgc.u-bordeaux2.fr
Paumard
P
P
Soubannier
V
V
Spannagel
C
C
Vaillier
J
J
Arselin
G
G
Graves
P V
PV
D025181:ATP Synthetase Complexes; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry
D003432:Cross-Linking Reagents; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); D006180:Proton-Translocating ATPases; D003545:Cysteine