Topography of the yeast ATP synthase F-0 sector

The interaction between the hydrophilic C-terminal part of subunit 4 (subun it b) and OSCP, which are two components of the connecting stalk of the yea st ATP synthase, was shown after reconstitution of the two over-expressed p roteins and by the two-hybrid method. The organization of a part of the F-o 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 reveal ed that the sulfhydryl groups were modified upon incubation of intact mitoc hondria. 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 additio n, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, th us demonstrating near-neighbor relationships of the two subunits and a loca tion of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space. (C) Societe francaise de biochimie et biologie molecu laire/Elsevier, Paris.