Figure 7.

Interaction of RIC9 with other subunits during complex assembly at the membrane. (A) Effect of RIC9 knockdown on mitochondrial inner membrane complexes. Mitochondrial extracts from uninduced (WT) or tetracycline-induced cultures of L. tropica 13–90 transformed with pGET(RIC9AS) (KD9) were subjected to BN electrophoresis, and the protein complexes visualized by Coomassie staining. The positions of RIC (R) and respiratory complexes V, IV and III in the WT extract are indicated; R^Δ9 and V^Δ9 are the subcomplexes in knockdown cells derived from RIC and complex V, respectively. (B) Wild-type RIC (wt) and knockdown complexes (R^ΔX) from cells deficient in subunit X, where X = subunit 6, 8A or 9. (C and D) In vitro assembly assay. Complexes from RIC9 (C) or RIC8A (D) knockdown cells were reconstituted in presence of liposomes and recombinant RIC9 or RIC8A, respectively. Where indicated, antibody (α) against a specific RIC subunit was present during the reconstitution reaction. Liposome-bound complexes were resolved by native (C) or denaturing (D) PAGE, western blotted and probed with anti-RIC9 (C) or anti-RIC8A (D) antiserum. (E) Interaction between RIC6 and RIC9 at the membrane. Recombinant RIC6 (0.2 μM) and RIC9 (0.2–0.6 μM) were incubated alone or in combination with liposomes, which were washed and the liposome-bound proteins resolved by native PAGE. Left, Coomassie stain. Duplicate lanes were blotted on nitrocellulose and probed with anti-RIC6 (middle) or anti-RIC9 (right) serum. (F) Hypothetical model of RIC, showing the nucleus-encoded subunits. Subunit numbering and stoichiometry are as in ref. (23). Subunits 1, 8A, 9 and 6 are highlighted in color. (G) Sequential assembly of subunits 6, 9 and 8A, indicating the steps blocked by specific antibodies. For clarity, the common subunits of the knockdown complexes (1, 4A, 5 and 8B, and the mitochondrion-encoded subunits 2, 4B and 7) are not shown.