Organisation of the yeast ATP synthase F-0: a study based on cysteine mutants, thiol modification and cross-linking reagents

A topological study of the yeast ATP synthase membranous domain was underta ken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagen esis of genes encoding ATP synthase subunits, The modification by non-perme ant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of t he Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in t he C-terminus of mutated subunit f demonstrated their location in the mitoc hondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobi functional reagents. Our data suggest interactions between the first transm embranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the en zyme and interacts with other ATP synthase complexes. (C) 2000 Elsevier Sci ence B.V. All rights reserved.