Environmental study of subunit i, a F-0 component of the yeast ATP synthase

The topology of subunit i, a component of the yeast FoF1-ATP synthase, was determined by the use of cysteine-substituted mutants. The N-in-C-out orien tation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 3-acetamido-4'-maleimidylstilbene-2,2'-disulf onic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl ox idation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a s toichiometry of 1 subunit i per complex. Crosslinked products between mutan t subunits i and proteins loosely bound to the FoF1-ATP synthase suggest th at subunit i is located at the periphery of the enzyme and interacts with p roteins of the inner mitochondrial membrane that are not involved in the st ructure of the yeast ATP synthase.