1. Blot : myc GRIM-19 com II myc BN-PAGE SDS-PAGE GRIM-19 GRIM19 Y143A GRIM-19 Y143D GRIM-19 G139V GRIM-19 G139R Supplementary Fig. 1 GRIM19 mutants (G139V, G139R, Y143D and Y143A) were transfected into 293T cells. Blue-Native PAGE was used to resolve mitochondrial respiratory chain complexes from these cells. Anti-myc antibody was used to detect transfected GRIM-19 and mutants. Blue-Native Western blot was used to check the amount of exogenous proteins inserted into mitochondrial complex I. Normal SDS-PAGE/Western blot was also conducted to check the total amount of exogenous protein present in mitochondria. Mitochondrial complex II was detected by anti-complex II 70 kDa subunit antibody.

2. Blot : HA GRIM-19 comII 70 kDa subunit HA BN PAGE SDS PAGE GRIM-19-HANDUFA9 1-100-HANDUFA9 1-80-HANDUFA9 1-60-HANDUFS3 1-100-HANDUFS3 1-80-HA NDUFS3 1-60-HA Supplementary Fig. 2 NDUFA9 mutants (1-100 and 1-80) and NDUFS3 mutant (1-100) can be assembled into mitochondrial complex I. WT-GRIM-19, NDUFA9 mutants (1-100, 1-80 and 1-60), and NDUFS3 mutants (1-100, 1-80 and 1-60) were transfected into 293T cells. The same procedure was conducted as in Supplementary Fig 1.

3. Complex I IP HA DN-GRIM-19 Supplementary Fig. 3 Mitochondrial complex I was immunoprecipated by complex I capture kit (MitoSciences (Eugene, OR, USA) according to manufacturer’s instructions. DN-GRIM-19-HA incorporated into complex I was immunoprecipated by HA-conjugated agarose beads. The immunoprecipated protein was eluted from the beads and subjected to SDS-PAGE. The gel was visualized by silver stain.