AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5

Abstract

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Keywords: AIFM1; MIA40-CHCHD4; NDUFS5; complex I; mitochondrial disulfide relay.

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

1. A

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

2. 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 with HEK293 cells. N = 4 biological replicates, an unpaired one‐sample two‐sided Student's t‐test was applied (P < 0.05, fold change >2).

3. 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 with 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).

4. 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 1 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.

5. 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 with HEK293 cells. N = 4 biological replicates, an unpaired one‐sample two‐sided Student's t‐test was applied (P < 0.05, fold change >2).

6. 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.

7. 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 wildtype cells, in line with a decrease in 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).

Figure 2. Absence of AIFM1 stalls complex I assembly likely due to impaired NDUFS5 insertion

1. A

   The modular assembly pathway of complex I. Simplified model as deduced from (Guerrero‐Castillo et al, 2017). The MIA40/CHCHD4 substrates NDUFA8, NDUFB7, NDUFB10, and NDUFS5 and their entry during complex I assembly are highlighted in shades of red and labeled omitting the leading “NDUF.” In AIFM1 knockout cells, levels of the P_D‐a, Q/P_P‐a, P_D‐b assembly intermediates, and the Q/P range were increased (green arrows). Levels of the P_P‐b, free N‐module, and P_P‐b/P_D‐a were not significantly changed (gray arrows). Levels of Q/P_P and complex I were decreased in AIFM1 knockout cells (red arrows). These findings indicate dramatic changes in complex I assembly in AIFM1 KO cells and point to specific stalling points (for details see Appendix Figs S3 and S4).

2. B

   Bar diagram showing a summary of the detailed analysis of the Q/P range using high‐resolution complexome profiling HEK293, AIFM1 knockout, and AIFM1 knockout complemented with AIFM1 or AIFM1‐HA (for details, see Appendix Fig S5). P_P‐b/P_D‐a and the assembly factors MCIA, TMEM70, and TMEM126A were enriched in this range. Their combined mass is, however, only half of the mass indicated by their migration in the Q/P range leading to the hypothesis that a dimer containing P_P‐b/P_D‐a and the assembly factors accumulates in AIFM1 knockout cells. N = 6 biological replicates, except AIFM1 KO + AIFM1 (N = 3); error bars indicate SD and an unpaired, two‐sided Student's t‐test was applied (*P < 0.05, **P < 0.01).

3. C

   Bar diagram showing amounts of NDUFB10 and NDUFS5 and the P_P‐b/P_D‐a assembly intermediate in HEK293, AIFM1 knockout, and AIFM1 knockout complemented with AIFM1 or AIFM1‐HA. Data are normalized to the wildtype assembly intermediates (WT = 1). NDUFS5 is missing from P_P‐b/P_D‐a although the module (and also NDUFB10) is present at normal levels. N = 6 biological replicates, except AIFM1 KO + AIFM1 (N = 3); error bars indicate SD and an unpaired, two‐sided Student's t‐test was applied (**P < 0.01, ***P < 0.001, ****P < 0.0001).

4. D

   Disulfide relay substrates in complex I. The structure of mammalian complex I (Agip et al, 2018) and the positioning of the four disulfide relay substrates NDUFA8, NDUFB7, NDUFB10, and NDUFS5 are shown as viewed from the IMS. Cartoon was prepared from PDB 6G2J using the PyMOL Molecular Graphics System, Version 2.0 (Schrödinger, LLC). Color code of submodules and individual proteins is the same throughout the manuscript. NDUFS5 is positioned below NDUFA8 in mature complex I.

5. E

   Model for altered complex I assembly in AIFM1 knockout cells. Complex I assembly stalls at the height of the P_P‐b/P_D‐a module due to the absence of NDUFS5. This leads to the accumulation of a dimer containing P_P‐b/P_D‐a.

Figure 3. NDUFS5 import and oxidation are impaired in AIFM1 knockout

1. A

   In organello import assay with NDUFS5, NDUFB7 and as controlling the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Nonimported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS–PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by the loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 and NDUFB7 are very similar. N = 3 replicates; error bars indicate SD.

2. B

   Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells, and AIFM1 knockout cells complemented with AIFM1‐HA were analyzed by pulse‐chase experiments coupled to redox state determination. To this end, cells were incubated with ³⁵S‐Met for 5 min (pulse). After removal of ³⁵S‐Met, cells were left in “cold” met for different chase times. Further oxidation was stopped by the addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24, and then, MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP) and analyzed by nonreducing SDS–PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5. Error bars indicate SD.

3. C

   Synthesis of NDUFS5 and NDUFB7 in wildtype and AIFM1 knockout cells. Cells were incubated with ³⁵S‐Met for 5 min (pulse), and then, MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP) and analyzed by nonreducing SDS–PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.

4. D

   Interaction between endogenous MIA40/CHCHD4 and its substrates in wildtype and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS–PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady‐state levels of these proteins are a consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band; orange arrow indicates a signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.

Figure 4. MIA40/CHCHD4 is present in a stable long‐lived complex with AIFM1

1. A

   Assessment of MIA40/CHCHD4 variant‐AIFM1 interaction. The indicated MIA40/CHCHD4‐Strep variants were affinity precipitated (AP) under native conditions after stopping thiol‐disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR, and MIA40/CHCHD4 by reducing SDS–PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40‐Strep. Both, wildtype MIA40/CHCHD4 and MTS‐MIA40/CHCHD4 but not MIA40/CHCHD4^Δ1–40 variants coprecipitated AIFM1. All mitochondria‐localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.

2. B, C

   Emetine‐chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4‐Strep (B) or AIFM1 knockout cells stably expressing AIFM1‐HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (Triton X‐100), and MIA40/CHCHD4‐Strep (B) or AIFM1‐HA (C) were precipitated. Precipitates were analyzed by reducing SDS–PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6–8 h (B, C), while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band. N = 3 biological replicates.

3. D, E

   Gel filtration analysis to assess the in vitro reconstituted AIFM1‐MIA40/CHCHD4 complex. Purified MIA40/CHCHD4 and AIFM1 were incubated in the presence of NADH and subjected to gel filtration analysis. Elution was monitored by following the absorption at 280 nm (D). Eluted fractions were subjected to SDS–PAGE and TCE staining (E). AIFM1 and full‐length MIA40/CHCHD4 migrate together indicating the formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and Δ1‐40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N‐terminal amino acids in MIA40 for interaction with AIFM1.

4. F

   Isothermal Titration Calorimetry (ITC) analysis of AIFM1 and MIA40/CHCHD4. The partners bind to each other with a K_D of approximately 0.18 μM and stoichiometry between AIFM1 and MIA40/CHCHD4 of 2 to 1. Data shown were normalized by the background signal of titrating MIA40/CHCHD4 into the buffer.

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

1. 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 the migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1‐HA. For a number of biological replicates and quantifications, see 5C.

2. B

   Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (Δ1‐40 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 a monomer. For a number of biological replicates and quantifications, see 5C.

3. C

   Quantification of 5A,B and Appendix Fig 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 in 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.

4. 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 the liver and kidney more abundant than MIA40/CHCHD4.

5. 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 a lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.

6. F

   Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated from 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.

7. 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.

Figure 6. In AIFM1 knockout cells, impaired OMM translocation of NDUFS5 results in its rapid proteasomal degradation

1. A

   Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells, and AIFM1 knockout cells complemented with AIFM1‐HA were analyzed by pulse‐chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 were not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation‐dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.

2. B

   Model. The AIFM1‐MIA40/CHCHD4 complex mediates proper oxidation‐dependent protein import and insertion of the four disulfide relay substrates into complex I. In the absence of AIFM1, monomeric soluble MIA40/CHCHD4 is still capable of importing many of its substrates. It fails, however, to accumulate NDUFS5. This protein is not properly oxidized and as a consequence, it becomes rapidly degraded by the proteasome. Lack of NDUFS5 results in stalling of complex I assembly and lowered levels of complex I.