Topography of the yeast ATP synthase F0 sector

The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCP, which are two components of the connecting stalk of the yeast ATP synthase, was shown after reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the F0 sector was 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 subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled 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.