ATP SYNTHASE COMPLEX - PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES

The catalytic sector, F-1, and the membrane sector, F-0, of the mitoch ondrial ATP synthase complex are joined together by a 45-Angstrom-long stalk. Knowledge of the composition and structure of the stalk is cru cial to investigating the mechanism of conformational energy transfer between F-0 and F-1. This paper reports on the near neighbor relations hips of the stalk subunits with one another and with the subunits of F -1 and F-0, as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particl es (SMP) and F-1-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-lin king from zero to 10 Angstrom. Cross-linked products were identified a fter gel electrophoresis of the particles and immunoblotting with subu nit-specific antibodies to the individual subunits alpha, beta gamma, delta, OSCP, F-6, A6L, a (subunit 6), b, c, and d. The results suggest ed that the two b subunits form the principal stem of the stalk to whi ch OSCP, d, and F-6 are bound independent of one another. Subunits b, OSCP, d, and F-6 cross-linked to alpha and/or beta, but not to gamma o r delta. The COOH-terminal half of A6L, which is extramembranous, cros slinked to d but not to any other stalk or F-1 subunit. No cross-links of subunits a and c with any stalk or F-1 subunits were detected. In F-1-deficient SMP, cross-linked b + b and d + F-6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. Th e addition of F-1 to F-1-deficient particles appeared to reverse these changes. Treatment of F-1-deficient particles with trypsin rapidly hy drolyzed away OSCP and F-6, fragmented b to membrane bound 18-, 12-, a nd 8-9-kDa antigenic fragments, which cross-linked to d and/or with on e another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near ne ighbor relationships of the stalk subunits with one another and with t he alpha and beta subunits at a level near the proximal end (bottom) o f F-1 and at the membrane-matrix interface are presented.