AIFM1 is a component of the mitochondrial disulfide relay that
drives complex I assembly through efficient import of NDUFS5 (2022)
Figure 5

Figure 5. The AIFM1-MIA40/CHCHD4 complex exists ubiquitously and serves in associating MIA40/CHCHD4 with the inner membrane.

A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. For number of biological replicates and quantifications, see 5C.

B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C.

C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.

D. Levels of AIFM1 and MIA40/CHCHD4 in mitochondria isolated from the indicated tissues. Ratio of AIFM and MIA40/CHCHD4 was formed and normalized to ratio in HEK293 cells (= 1). AIFM1 is in liver and kidney more abundant than MIA40/CHCHD4.

E. Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells. Experiment was performed as described in (A). AIFM1 and MIA40/CHCHD4 co-migrate in all samples. Excess AIFM1 migrates at lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.

F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1.

G. Model. Almost all cellular MIA40/CHCHD4 exists in a permanent complex with AIFM1. ALR is not part of the complex. Being in this complex renders MIA40/CHCHD4 membrane-associated. In AIFM1 knockout cells, MIA40/CHCHD4 is present as a soluble monomeric protein.