AIFM1 is a component of the mitochondrial disulfide relay that
drives complex I assembly through efficient import of NDUFS5 (2022)
Figure 1

Figure 1. AIFM1 knockout results in decreased complex I levels despite normal amounts of MIA40/CHCHD4.

A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells.

B. Proteomic analysis comparing MIA40/CHCHD4 levels in HEK293 and AIFM1 knockout mitochondria. Mitochondria from the SILAC labeled cells were subjected to proteomic analyses (Dataset EV1). MIA40/CHCHD4 levels are unchanged in AIFM1 knockout cells compared to HEK293 cells. N = 4 biological replicates, an unpaired one-sample two-sided Student's t-test was applied (p<0.05, fold change >2).

C. Proteomic analysis comparing MIA40/CHCHD4 substrate levels in HEK293 and AIFM1 knockout mitochondria (same as B.). Mitochondria from the SILAC labeled cells were subjected to proteomic analyses (Dataset EV1). Levels of many MIA40/CHCHD4 substrates were significantly reduced in AIFM1 knockout cells compared to HEK293 cells. Notably, this appears to be selective as other MIA40/CHCHD4 substrates were not or only slightly affected by AIFM1 deletion. N = 4 biological replicates, an unpaired one-sample two-sided Student's t-test was applied (p<0.05, fold change >2).

D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates. Gray box indicates subunits of complex I.

E. Proteomic analysis comparing respiratory chain protein levels in HEK293 and AIFM1 knockout mitochondria (same as B.). Mitochondria from the SILAC labeled cells were subjected to proteomic analyses (Dataset EV1). Mainly levels of complex I subunits were significantly reduced in AIFM1 knockout cells compared to HEK293 cells. N = 4 biological replicates, an unpaired one-sample two-sided Student's t-test was applied (p<0.05, fold change >2).

F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates

G. Activity analyses of respiratory chain complex I in HEK293, AIFM1 knockout and AIFM1 knockout complemented with AIFM1-HA or AIFM1. Activity of complex I was assessed in broken mitochondrial membranes and normalized to the activity of complex II. In AIFM1 knockout cells, complex I shows a significantly decreased activity to about 30-40% of wild type cells, in line with a decrease of total complex I levels to about 30-40%. Thus, the specific activity of still assembled complex I in AIFM1 knockout cells is unchanged. Complementation of AIFM1 knockout cells with both, AIFM1 or AIFM1-HA rescues activity of complex I. N = 7 biological replicates; error bars indicate SD and an unpaired, two-sided Student's t-test was applied (** = p<0.01, **** = p<0.0001).