FIGURE 4.

Purification of partially assembled F_OF₁ of mutants Δa and Δδ by affinity chromatography in comparison with wild type. A and C, comparison of membranes and purified F_OF₁ complexes. B and D, detection of the different F_OF₁ subunits to verify their presence in the complexes purified. DK8 transformed with pBWU13 derivatives was grown as described in the legend of Fig. 2. Membranes were prepared and solubilized, and F_OF₁ complexes were purified by affinity chromatography via a His₆ tag (A and B) present N-terminal to subunit β (WT, pBH7; Δa, pBH55.β-His; Δδ, pBH56.β-His) or a His₁₂ tag (C and D) fused to the N terminus of subunit a (WT, pBH1; Δδ, pBH56.a-His12). Membranes and purified F_OF₁ were analyzed by immunoblotting. S, molecular mass standard; C, subunits a and b detected in membranes of DK8/pBWU13.