Skip to main content
. Author manuscript; available in PMC: 2018 Jan 31.
Published in final edited form as: Cell Rep. 2017 Jul 5;20(1):264–278. doi: 10.1016/j.celrep.2017.06.015

Figure 3. Proteomic Analyses And Immunoblotting Identify Assembly Intermediates Of CI.

Figure 3

(A) Schematic of CI showing the three modules of the enzyme. The NADH Dehydrogenase module (N module) is located at the tip of the matrix arm, and is the site of NADH oxidation. Situated between the N module and the membrane arm, is the Q module, which is responsible for Ubiquinone reduction. The proton-conducting P module is in the membrane arm.

(B) The current model of CI assembly in mammalian systems (reviewed in (Vartak et al., 2014). The assembly process begins with the formation of an assembly intermediate containing NDUFS2 and NDUFS3, which combines with NDUFS7 and NDUFS8. The subcomplex of NDUFS2, NDUFS3, NDUFS7 and NDUFS8 ultimately combines with ND1 to form the ~315 kDa assembly intermediate that is anchored to the membrane. The ~315 kDa subcomplex (also called the Q module) combines with an independently-formed ~370 kDa assembly intermediate to form an ~550 kDa assembly intermediate. This assembly intermediate which consists of the Q module and part of the P module grows by the addition of more subunits to form the ~815 kDa assembly intermediate, via mechanisms that are very poorly defined. The ~815 kDa assembly intermediate now consists of the complete Q and P modules. Finally, the N module is added to produce the 950kDa fully-assembled complex. Assembly factors or chaperones that assist in this process, but are not present in the fully assembled complex, have been omitted for clarity.

(C) Western blot of samples obtained from thoraxes from pupae aged between 2 and 4 days after pupariation, and of flies from 0.5 hours to 48 hours post-eclosure to detect the assembly intermediates, fully assembled CI, and a supercomplex containing complex I (supCI) after BN-PAGE. The anti-NDUFS3 antibody strongly detects CI and supCI; and weakly detects the ~315 kDa, ~550 kDa and ~815 kDa assembly intermediates after a short exposure. However, after a longer exposure, the ~315 and ~550 kDa assembly intermediates can clearly be seen. In the right panel, the membrane was stripped and re-probed with anti-NDI. Anti-ND1 detects the ~315 kDa and ~550 kDa assembly intermediates, and a very faint band corresponding to CI. Note that in all 4 panels there is a general increase in accumulation of assembly intermediates, holoenzyme and CI-containing supercomplex with time. The CV antibody (anti- ATPsynß) was used as a loading control because commercially-available anti-CII antibodies we tested did not cross-react with Drosophila CII.

(D) Proteomic analyses of assembly intermediates that form in the native gel sized between ~50 kDa and ~350 kDa. See Table S3 for all the peptides identified.