In earlier work, we [McCormick, K. A., et al. (1993) J. Biol. Chem. 268, 24683-24691] observed that mutations at Ala-79 of the b subunit affect assembly of F1F0 ATP synthase. Polypeptides modeled on the soluble portion of the b subunit (b(sol)) with substitutions at the position corresponding to Ala-79 have been used to investigate secondary structure and dimerization of the b subunit. Circular dichroism spectra and chymotrypsin digestion experiments suggested that the recombinant polypeptides with Ala-79 substitutions assumed conformations similar to the b(sol) polypeptide. However, cross-linking studies of the Ala-79 substitution b(sol) polypeptides revealed defects in dimerization. The efficiency of dimer formation appeared to be related to the capacity of the altered b(sol) polypeptides for competing with F1-ATPase for binding to F1-depleted membrane vesicles. Ala- 79 substitution polypeptides displaying limited dimerization, such as b(sol) (Ala-79→Leu), were shown to elute with F1-ATPase during size exclusion chromatography, suggesting a specific interaction. Sedimentation equilibrium studies indicated that 8% of the b(sol) (Ala-79→Leu) polypeptide was in the form of a 30.6 kDa dimer and 92% a 15.3 kDa monomer. When the dimer concentration of b(sol) (Ala-79-Leu) was normalized to the concentration of b(sol), both had virtually identical capacities for competing with F1- depleted membrane vesicles for binding F1-ATPase. The result indicated that the amount of dimer formed is directly proportional to its ability to bind F1-ATPase. This suggests that formation of the b subunit dimer may be a necessary step preceding F1-ATPase binding in the assembly of the enzyme complex.
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