Figure 1. C9orf72 is an Inner Mitochondrial Membrane Binding Protein.
(A) Isolated mitochondria (Mito) from WT and C9KO MEFs were subjected to digestion with proteinase K (PK). C9orf72, an IMM protein TIM23, a matrix protein NDUFB9, and OMM proteins TOM70 and TOM20 were used as controls.
(B) Mitochondria isolated from WT and C9KO MEFs were either sonicated or treated with sodium carbonate to extract peripheral membrane-binding proteins from the pellet fraction (P) to the supernatant fraction (S).
(C) Representative confocal images and quantification of colocalization of endogenous C9orf72 and TIM23 in WT and C9KO MEFs (n = 40 cells). Scale bars, 10 μm.
(D) C9orf72 immuno-electron microscopy (EM) analysis of WT and C9KO mitochondria isolated from mouse brain cortex tissues (n = 14 images). Red arrows point to immunogold-labeled C9orf72. Scale bars, 100nm.
(E and F) The gel-shift AMS-modification assay was used to measure the oxidation states of C9orf72 in cytosolic and mitochondrial fractions of HEK293 cells. R: Reduced. O(p): Partially oxidized. O: Oxidized. The oxidation states of native C9orf72 are quantified (n = 4) (F).
(G) HEK293 cells with knockdown of CHCHD4 or AIFM1 were subjected to subcellular fractionation and immunoblotting of indicated proteins (n = 4).
(H) Representative autoradiographs and quantitative analysis of [35S]C9orf72 imported into mitochondria isolated from HEK293 cells treated with the indicated siRNAs and proteinase K (n = 3).
(I) Kinetic analysis of the C9orf72 import reaction as shown in (H). A representative gel image and quantification are shown (n = 3).
Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S1 and S2, and Table S1.