Topography of the yeast ATP synthase F0 sector by using cysteine substitution mutants. Cross-linkings between subunits 4, 6, and f

Abstract

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.