Figure 5. Identification Of An ~700 kDa Assembly Intermediate Of CI In Drosophila.

(A) Top Panel: Immunoblots of samples obtained from wildtype and mhc>dNDUFS5^RNAi thoraxes of flies aged for 6 hours after eclosure depicting co-migration of the ~700 kDa intermediate and CV. In the left and middle panels, anti-NDUFS3 antibodies detect the fully assembled CI, the ~700 kDa subcomplex, as well as other assembly intermediates in dNDUFS5^RNAi thoraxes. Note that in the middle panel, the region of the membrane just below CI was cut and imaged. In the right panel, anti-ATPsynβ detects the CV monomer (700kDa) and dimer as shown. Lower Panel: Mitochondrial protein complexes from wildtype and mhc>dNDUFS5^RNAi thoraxes were resolved by BN-PAGE and the region corresponding to the ~700 kDa assembly intermediate (i.e. CV, demarcated) was cut out, subjected to tryptic digestion, and analyzed by label-free quantitative LC-MS/MS.

(B) Immunoblots from samples obtained after 6 hours, 12 hours and 24 hours post eclosure from thoraxes where NDUFS1, NDUFS3, NDUFS5 and NDUFV1 were knocked down as a result of transgenic RNAi exression. Note that the ~815 kDa assembly intermediate accumulates as a result of disruption of NDUFS1 and NDUFV1, and the ~700 kDa assembly intermediate stalls and accumulates in NDUFS5 mutants at all time points. Importantly, upon prolonged exposure of the immunoblot, a band corresponding to the ~700 kDa assembly intermediate can also be observed in wild-type samples (denoted with the * in the lower panel), which confirms that it is an authentic, albeit transient assembly intermediate.

(C) The accumulation of the ~815 kDa assembly intermediate was significantly attenuated in mhc>dNDUFS5^RNAi,dNDUFS1^RNAi thoraxes relative to mhc>dNDUFS1^RNAi thoraxes; instead there is an accumulation of the ~700 kDa assembly intermediate. Similar results were obtained when samples from mhc>dNDUFS5^RNAi,dNDUFV1^RNAi thoraxes were compared to samples from mhc>dNDUFV1^RNAi thoraxes.

(D) Proteomic changes in the gel slice sample from wildtype and mhc>dNDUFS5^RNAi thoraxes corresponding to the ~700 kDa assembly intermediate. Relative protein abundance among biological samples is expressed by spectral counts on a log scale. Several CI subunits and CIAFs, most notably components of the MCIA complex are upregulated in the ~700 kDa assembly intermediate. However, the amount of dNDUFA10 (denoted with an asterisk) is reduced in mhc>dNDUFS5^RNAi thoraxes relative to wild type. See Table S4 for all the peptides identified