Abstract
The catalytic sector, F1, and the membrane sector, F0, of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F0 and F1. This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits of F1 and F0, as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particles (SMP) and F1-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-linking from zero to 10 A. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits alpha, beta, gamma, delta, OSCP, F6, A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F6 are bound independent of one another. Subunits b, OSCP, d, and F6 cross-linked to alpha and/or beta, but not to gamma or delta. The COOH-terminal half of A6L, which is extramembranous, cross-linked to d but not to any other stalk or F1 subunit. No cross-links of subunits a and c with any stalk or F1 subunits were detected. In F1-deficient SMP, cross-linked b+b and d+F6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. The addition of F1 to F1-deficient particles appeared to reverse these changes. Treatment of F1-deficient particles with trypsin rapidly hydrolyzed away OSCP and F6, fragmented b to membrane-bound 18-, 12-, and 8-9-kDa antigenic fragments, which cross-linked to d and/or with one another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near neighbor relationships of the stalk subunits with one another and with the alpha and beta subunits at a level near the proximal end (bottom) of F1 and at the membrane-matrix interface are presented.
Highlights
The catalytic sector, F 10 and the membrane sector, F 0, of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk
Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F 0 and F 1 This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits ofF1 and F0, as revealed by crosslinking experiments
Cross-linking of submitochondrial particles (SMP) with Disuccinimidyl tartarate (DST) or ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)-Even though our preparations of the ATP synthase complex display the same activities and inhibitor-response properties as the ATP synthase complex in SMP, we chose to carry out our studies of the near neighbor relationships of the ATP synthase subunits primarily on well coupled SMP lest the purification process, which involves the use detergents, might have introduced some structural changes in the isolated enzyme
Summary
The catalytic sector, F 10 and the membrane sector, F 0 , of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits a, p, y, o, OSCP, F 6 , A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F6 are bound independent cross-linked of to o a ne anot and/or he p, r. B. terminal half of A6L, which is extramembranous, crosslinked to d but not to any other stalk or F 1 subunit. In F 1-deficient SMP, crosslinked b + b and d + F6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. Trypsin removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d.
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