TOPOGRAPHY OF THE YEAST ATP SYNTHASE F-0 SECTOR BY USING CYSTEINE SUBSTITUTION MUTANTS - CROSS-LINKINGS BETWEEN SUBUNIT-4, SUBUNIT-6, AND SUBUNIT-F

The arrangement of the N-terminal part of subunit 4 (subunit b) has be en studied by the use of mutants containing cysteine residues in a loo p connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunit 4 by the fluorescent probe -(7-(dime thylamino)4-methyl-3-coumarinyl)maleimide revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addit ion, the nonpermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilb ene-2,2'-disulfonic acid prevented the 3-(N-maleimidylpropionyl)biocyt in labeling of subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the pro ximity of subunits 4 and f. In addition a disulfide bridge between sub unit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighb or relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane spa ce.