Fig 2: HIGD-1B delays OPA1 cleavage. (A) AC16 cells were transfected with empty or HIGD-1B-Flag vector, then incubated under hypoxic conditions. (B) Transfected AC16 cells were treated with CCCP. Total cell lysates were subjected to western blot analysis using an anti-OPA1 and Hsp60 antibodies. (C) AC16 cells were transfected with HIGD-1B-Flag or empty vector, then cultured under normoxic or hypoxic conditions. Western blotting was performed to detect HIGD-1B expression in each group. The cells were visualized under a fluorescent microscope following staining with MitoTracker. Scale bar, 10 µm. (D) AC16 cells were transfected with empty vector or HIGD-1B-Flag, then incubated under hypoxic conditions. (E) Transfected AC16 cells were treated with 10–50 µM CCCP for 1 h. Total viable cells were examined using trypan blue exclusion. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; HIGD-1B, HIG1 hypoxia inducible domain family member 1B; OPA1, optic atrophy 1; Hsp, heat shock protein; L, long; S, short.

Fig 3: HIGD-1B interacts with OPA1. The 293T cells were co-transfected with plasmids. Cell lysates were immunoprecipitated with (A) anti-Flag and (B) HIGD-1B antibody or control IgG, followed by western blot analysis. (C) SDS-PAGE for the purified GST-tagged HIGD-1B, Myc-tagged and GST proteins. Purified GST-HIGD-1B or GST protein bound to glutathione-Sepharose 4B was incubated with purified Myc-OPA1. Bound complexes were analyzed via western blot analysis using anti-GST or anti-Myc. GST, glutathione S-transferase; HIGD-1B, HIG1 hypoxia inducible domain family member 1B; OPA1, optic atrophy 1; L, long; S, short.

Fig 4: OPA1 high molecular oligomeric complexes are increased, and the short versus long forms are altered in SLC25A46 loss-of-function cells.(A) Two-dimensional electrophoresis (BN-PAGE/SDS–PAGE) analysis of mitochondria from the control, knock-out, and re-expression of WT-SLC25A46 and the pathogenic variant p.T142I. Membranes were immunoblotted for the indicated proteins. OPA1 complexes are highlighted: green boxes indicate the high molecular weight complexes (>600 kD), blue boxes indicate the molecular weight complexes of 200–400 kD, and red boxes indicate the monomeric OPA1 forms. (A, B) Quantification of the high molecular weight complexes (>600 kD) of OPA1 (indicated in the green boxes in (A)) relative to the total signal (n = 3). (C, D) Quantification of the relative proportions of the long versus short forms of OPA1 forms in the high molecular weight complexes (>200 kD) as indicated in the example (D) showing the longer forms of the higher complexes in the yellow box and the shorter forms in the purple box (n = 3).

Fig 5: MFN2 oligomerization is influenced by SLC25A46 and OPA1.(A) Analysis of MFN2 and OPA1 oligomerization (by BN-PAGE and SDS–PAGE) of isolated mitochondria from control and SLC25A46 knock-out fibroblasts in which MFN2 or OPA1 was knocked down by siRNA constructs for 6 d. siRNA-mediated knock-down of SLC25A46 in control cells is also shown. (B) Immunofluorescence analysis of fibroblasts after 6 d of siRNA-mediated knock-down of MFN2 and OPA1 in control and SLC25A46 knock-out fibroblasts. Mitochondria were decorated with anti-PRDX3 in white and DAPI in blue. Scale bar: 10 μm.