The second stalk of the yeast ATP synthase complex: identification of subunits showing cross-links with known positions of subunit 4 (subunit b).
A component of the stator of the yeast ATP synthase (subunit 4 or b) showed many cross-linked products with the homobifunctional reagent dithiobis[succinimidyl propionate], which reacts with the amino group of lysine residues. The positions in subunit 4 that were involved in the cross-linkings were determined by using cysteine-generated mutants constructed by site-directed mutagenesis of ATP4. Cross-linking experiments with the heterobifunctional reagent p-azidophenacyl bromide, which has a spacer arm of 9 A, were performed with mitochondria and crude Triton X-100 extracts containing the solubilized enzyme. Substitution of lysine residues by cysteine residues in the hydrophilic C-terminal part of subunit 4 allowed cross-links with subunit h from C98 and with subunit d from C141, C143, and C151. OSCP was cross-linked from C174 and C209. A cross-linked product, 4+beta, was also obtained from C174. It is concluded that the C-terminus of subunit 4 is distant from the membrane surface and close to F(1) and OSCP. The N-terminal part of subunit 4 is close to subunit g, as demonstrated by the identification of a cross-linked product involving subunit g and the cysteine residues 7 or 14 of subunit 4.
Soubannier
V
V
Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Segalen, Bordeaux 2, 1 rue Camille Saint-Saëns 33077 Bordeaux Cedex, France.
Rusconi
F
F
Vaillier
J
J
Arselin
G
G
Chaignepain
S
S
Graves
P V
PV
Schmitter
J M
JM
Zhang
J L
JL
Mueller
D
D
Velours
J
J
D025181:ATP Synthetase Complexes; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D007202:Indicators and Reagents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D013388:Succinimides; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); C011240:dithiobis(succinimidylpropionate)