Substructure of F₁-ATPase (BF₁ factor) from Micrococcus lysodeikticus

Summary

Dimethyl suberimidate and dithiobis (succinimidyl propionate) have been used to explore the nearest neighbor relationship of the subunits (α, β, γ and δ by decreasing molecular weight) of F₁-ATPase or BF₁ factor of Micrococcus lysodeikticus. Cross-linking with the two diimido esters inhibited the ATPase activity but this inhibition never exceeded 50% of the initial value. The cross-linking pattern of this BF₁ factor, as revealed by sodium dodecyl sulfate gel electrophoresis, shows a relative low proportion of high molecular weight aggregates which move slowly than the heaviest subunit (α). They are resolved as three components of molecular weights 200,000, 130,000 and 100,000 in 5% acrylamide gels, plus an additional component (mol. wt 80,000) identified in 10% acrylamide gels. The other aggregate bands represent cross-linking products of the smaller subunits (γ and δ) that may travel to the conventional position of the heavier subunits.

The subunit composition of the aggregate bands has been determined through the reversion of dithiobis (succinimidyl propionate) cross-linking of the BF₁ factor by dithiothreitol and analysis in second dimension by gel electrophoresis. The results indicate that γ subunit can cross-link with itself and with each of the other subunits except β. The α subunit is also able to cross-link with itself and with the other subunits although to a minor extent than γ, and that δ₂ aggregates are present. These results represent a specific pattern of cross-linking for this BF₁ factor as compared to other F₁ coupling factors. It suggests a certain asymmetry in the spatial organization of the major subunits of M. lysodeikticus F₁-ATPase where the γ subunit must play a central role. A subunit stoichiometry α₃ β₃ γ₂ δ₂ is proposed for whole F₁-ATPase which leads to a molecular weight 440,000 consistent with the 430,000 value estimated by sedimentation equilibrium at low speed. A tentative structural model of M. lysodeikticus BF₁ factor is derived from these data. The significance of the results in relation to the possible generalization of the molecular architecture of F₁ factors is discussed.