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. 2019 Nov 4;8:e48828. doi: 10.7554/eLife.48828

Figure 1. Cells lacking hTim8a have no defects in the TIM22, TIM23 or carrier biogenesis pathway.

(A–E) Mitochondria were isolated from control, (A) hTim9MUT HEK293 cells, (B) hTim8aKO HEK293 cells, (C) hTim8aMUT SH-SY5Y cells, (D) hTim8bKO HEK293 cells, or (E) hTim8bKO SH-SY5Y cells prior to solubilisation in 1% digitonin-containing buffer. Mitochondrial lysates were subjected to Blue-Native electrophoresis prior to immunoblotting using the indicated antibodies. (F–H) [35S]-hTim23, [35S]-hTim22 or [35S]-GC1 were incubated with mitochondria isolated from control and hTim8aKO or hTim8bKO) HEK293 cells for the indicated time in the absence or presence of a mitochondrial membrane potential (ΔΨ) prior to Proteinase K treatment. Samples were separated by SDS-PAGE or solubilised in 1% digitonin-containing buffer and separated by BN-PAGE and visualised using autoradiography. (I) Mitochondria isolated from control SH-SY5Y and hTim8aMUT SH cells were incubated with [35S]-hTim23 or [35S]-hTim22 for 60 min in the presence or absence of membrane potential (ΔΨ) before Proteinase K treatment. Mitochondria were reisolated and solubilised in digitonin prior to BN-PAGE and subsequent immunoblotting.

Figure 1.

Figure 1—figure supplement 1. Generation of CRISPR/Cas9 genome-edited cell lines.

Figure 1—figure supplement 1.

(A–E) Schematic representation of the CRISPR/Cas9 editing system used to generate (A) hTim9MUT (MUT = mutant) in Flp-In T-REx HEK293 cells, (B) hTim8aKO (KO = knock out) in Flp-In T-REx HEK293 cells, (C) hTim8aMUT (MUT = mutant) in SH-SY5Y cells (D) hTim8bKO in Flp-In T-REx HEK293 cells or (E) hTim8bKO (KO = knock out) in SH-SY5Y cells.
Figure 1—figure supplement 2. Loss of hTim8a or hTim8b has no defect on TIM or carrier biogenesis.

Figure 1—figure supplement 2.

(A) Mitochondria isolated from control and hTim9MUT, hTim8aKO, hTim8aMUT SH or hTim8bKO SH cells, were subjected to SDS-PAGE analysis and immunoblotted using the indicated antibodies. (B) Relative abundance of TIM22 complex and TIM23 complex in control and hTim8aMUT SH-SY5Y mitochondria (refer to the BN-PAGE/western blot analysis in Figure 1C) were quantified and tabulated as mean ± SD (n = 3).