Fig 1.
MIC13 is identified as MICOS subunit using complexome profiling.
(A) The graph shows the normalized occurrence of the proteins which cocluster with the MIC60 and other MICOS components in control HEK293 cells. Using this complexome profile, MIC13 is identified as MICOS component. (B) Endogenous MIC13, MIC60 and MIC27 antibodies were used for coimmunoprecipitation. Preimmuneserum (PIS) was used as the control. Endogenous MIC13 could pull down MIC60 and reciprocally in control 143B cells, MIC60 and MIC27 can pull down MIC13, indicating that MIC13 is the part of the MICOS complex. (C) Sequence alignment of MIC13 shows that it is conserved from Caenorhabditis elegans to higher eukaryotes.
Fig 2.
MIC13 localizes to inner mitochondrial membrane.
(A) Representative images of mitochondria (marked by mito-GFP, green) and endogenous MIC13 (using MIC13 antibody, red) or MIC13-FLAG (marked by anti-FLAG) in control 143B cells. Merge shows the colocalization of mitochondria and MIC13. Scale bar 10μm. (B) Isolated mitochondria of control 143B cells were swelled by osmotic shock (OS) and then treated or untreated with Proteinase K (PK). Triton-x-100 (Tx100) was used to permeabilize all the membranes. TOM20, TIM23 and TFAM were used for outer membrane, inner membrane and matrix marker respectively.
Fig 3.
MIC13 KO cells have no crista junctions.
(A) Immunoblot of MIC13 showing the complete loss of the protein in the knockout cell lines. (B) Representative EM images of control and MIC13 KO mitochondria (we analyzed approx. 40 to 50 mitochondria of each MIC13 KO cell line (N = 2). There is a complete loss of CJs in MIC13 KO cells whereas in control cells 1 to 5 CJs were observed in nearly all sections (40 to 50 mitochondria, N = 2). (C) Immunoblot of MIC13 in HeLa cells where MIC13 is depleted using SiRNA. (D) Representative EM images of control and MIC13 siRNA mitochondria.
Fig 4.
MIC13 knockout results in smaller but assembled MICOS complexes.
(A) Protein complexes were isolated by blue-native electrophoresis (BNE) on 3 to 18% acrylamide gradient gels immuno-decorated against MIC60, subunit coxVIa/b of complex IV and ATP synthase was shown on blot to demonstrate equal loading of samples and no effect. In control HEK293 cells, MICOS is detected around 500 KDa. Deletion of MIC13 leads to smaller MICOS complex. (B) Complexome profiling of the MICOS complex in MIC13 KO cells demonstrate the smaller MICOS complex (subcomplex) comprises of MIC60, MIC19 and MIC25. (C) Immnuoblot showing the steady state levels of various MICOS components in MIC13 KO cells as well as cells treated with MIC13 siRNA. There is reduction of MIC10, MIC27 and MIC26 upon deletion of MIC13. (D) Immunoblot from the cells depleted of MIC10 and probed for MIC13 and MIC60.
Fig 5.
Stability and assembly of respiratory chain complexes are unchanged upon MIC13 depletion.
(A) Immunoblot showing the steady state levels of various subunits of RC. (B) Blue-native electrophoresis of protein complexes stained with coomassie to detect major respiratory chain complexes in control and MIC13 KO cells. (C) Oxygen consumption rates of control and MIC13 KO1 cells are plotted as histogram (Mean±SE, N = 6). The basal, leak, ETS (electron transport system capacity or maximum respiration) and ROX (residual respiration) are shown for control and MIC13 KO1 cells. **, p value < 0.01. (D) Oxygen consumption rates measured in digitonin permeabilized cells after addition of indicated respiratory substrates and inhibitors. Basal, malate/glutamate (Complex I), ADP, succinate (Complex II/III), and ascorbate/TMPD (Complex IV) respiration are represented as box plots on which the median and 75/25 percentiles is plotted. In addition, all data values from seven individual experiments are plotted (Δ = Exp.1, × = Exp. 2, ◊ = Exp. 3, – = Exp. 4, □ = Exp. 5, ○ = Exp. 6, + = Exp. 7). *, p value < 0.05; ***, p value < 0.001.