FIGURE 3.

Ancient accessory subunits of CI. (A) NDUFS4 (berry), (B) NDUFS6 (plum) and (C) NDUFA12 (gold) are shown as colored surfaces on CI, the core subunits are shown as colored cartoons and other accessory subunits are shown as grey cartoons. Shin view of (D) mammalian (O. aries PDB: 6ZKC throughout), (E) E. coli (PDB: 7NZ1 throughout) and (F) T. thermophilus (PDB: 4HEA throughout) CI structures shown as transparent surfaces. NDUFS4 and the NDUFS1 loops (orange) that differ between the structures are shown as cartoons. (G) Calf view of Y. lipolytica CI structure (PDB ID: 6YJ4) shown as transparent surfaces with NDUFS6 (NUMM) and NDUFA12 (N7BM) shown as cartoons with the Zn²⁺ ion shown as atomic sphere and coordinating residues shown as sticks colored by element. Calf view of (H) mammalian, (I) E. coli and (J) T. thermophilus CI structures shown as transparent surfaces with NDUFS6 and the NDUFS8 (sea green) loops that differ between the structures labeled and shown as cartoons. The FeS clusters and Zn²⁺ ions are shown as atomic spheres and colored by element. (K) Structural differences between assembly factor NDUFAF2 and accessory subunit NDUFA12. Calf view of CI showing assembly factor NDUFAF2 from Y. lipolytica (PDB: 6RFQ) superposed on NDUFA12 (N7BM) (PDB: 6RFR). The transparent surface of CI is shown with NDUFA12 as cartoon in gold and NDUFAF2 shown as cartoon colored in dark olive. (L) Lipid binding around the Q-tunnel. Looking up from the Y. lipolytica (PDB: 6YJ4) CI heel showing the accessory subunits NDUFA9 (NUEM) (lavender), NDUFA12 (N7BM) and core subunit NDUFS7 (NUKM) (tea green) as cartoon. Other subunits are shown as transparent surfaces. Lipids are shown as spheres colored by element. (M) The interface between NDUFS4 (NUYM), NDUFA12 (N7BM) and NDUFS6 (NUMM) of Y. lipolytica CI (PDB: 6RFR). NDUFS4, NDUFA12 and NDUFS6 are shown as surfaces and all other CI subunits are shown as cartoon. (N) Clash between NDUFS6 and NDUFAF2. Y. lipolytica NDUFAF2 (PDB: 6RFQ) superposed on to NDUFA12 (N7BM) (PDB: 6RFR). NDUFS6 (NUMM) and NDUFAF2 are shown as cartoon. NDUFA12 (N7BM) is not shown for clarity. (O) Schematic diagram showing the role of ancient accessory subunits in the “checkpoint” hypothesis of the CI assembly pathway. NDUFS4 binds to CI Q/P subassembly bound to NDUFAF2 and disengages the C-terminal unstructured region of NDUFAF2. Addition of NDUFS6 and N-module to the subassembly results in the release of NDUFAF2 and frees up the NDUFA12 binding site. NDUFA12 binds to the subassembly at the site of NDUFAF2 and completes the assembly. Subunits are colored as in Figure 1 throughout unless stated otherwise. NDUFS4: berry, NDUFS6: plum, NDUFA12: gold, NDUFS1: orange, NDUFS8: sea green, NDUFAF2: dark olive, NDUFA9: lavender, NDUFS7: tea green.