Topography of the yeast ATP synthase F0 sector by using cysteine substitution mutants. Cross-linkings between subunits 4, 6, and f.
The arrangement of the N-terminal part of subunit 4 (subunit b) has been studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunit 4 by the fluorescent probe N-(7-(dimethylamino)-4-methyl-3-coumarinyl)maleimide revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, the nonpermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid prevented the 3-(N-maleimidylpropionyl)biocytin labeling of subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space.
Spannagel
C
C
Institut de Biochimie et Génétique Cellulaires du CNRS, Université de Bordeaux II, 1 Rue Camille Saint Saëns, 33077 Bordeaux Cedex, France.
Vaillier
J
J
Chaignepain
S
S
Velours
J
J
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics; D005456:Fluorescent Dyes; D008301:Maleimides; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013439:Sulfhydryl Reagents
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008301:Maleimides; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D006180:Proton-Translocating ATPases; D003545:Cysteine