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. Author manuscript; available in PMC: 2022 Nov 18.
Published in final edited form as: Science. 2022 Oct 20;378(6617):317–322. doi: 10.1126/science.add1856

Fig. 4. MTCH2 is a master regulator of outer membrane function.

Fig. 4.

(A) (Top) SLC25 transporters are composed of three sets of two TMDs (six total). The location of the characteristic Px[D/E]xx[K/R] motif within a single SLC25 repeat is indicated. (Bottom) Sequence alignment of helices 1, 3, and 5 (with starting residues indicated) from two canonical inner membrane SLC25 transporters (ADT1, UCP1) and two diverged outer membrane SLC25 transporters (MTCH1, MTCH2), with residues from the Px[D/E]xx[K/R] motif highlighted. (B) Flow cytometry analysis of OMP25-GFP11 integration into the outer membrane using the reporter assay described in Fig. 1B. MTCH1 was depleted by transient knockout in either wild type (wt) or MTCH2 knock out (ko) cell lines. (C) (Top) AlphaFold2 predicted model of MTCH2 highlighting conserved polar and charged residues within the bilayer colored based on their effects on OMP25 shown below. (Bottom) using the reporter strategy shown in Fig. 1B, the indicated MTCH2 mutants, which alter the electrostatic potential of its TMDs, were tested for their effect on the indicated reporters (fig. S20). Depicted is a heat map summarizing the stimulation of each mutant relative to wild type MTCH2 on biogenesis of MTCH2 independent (MICU1, LACTB1, TIM9A, USP30) and dependent (MAVS, OMP25, FUNDC1) substrates. (D) Cell lines expressing GFP1–10 in the ER lumen were used to monitor mislocalization to the ER of mitochondrial TAs fused to a C-terminal GFP11. Table summarizing the analysis when either MTCH2 is depleted or overexpressed (data in fig. S20A, fig. S21, and fig. S22). (E) K562 cells expressing varying levels of MTCH2 or inactive (D189R) or hyperactive MTCH2 mutants (E127R or K25E; Fig. 4C) were treated with the chemotherapeutic imatinib mesylate (IB; 1 μM) or carrier (DMSO) for 72 hours. Apoptosis was assessed by staining with Annexin-V-FITC and analyzed by flow cytometry. Shown are representative dot plots displaying the fraction of apoptotic cells upon IB treatment expressing wt MTCH2 compared to in inactive (D189R) or hyperactive mutant (K25E) (Top) as well as a summary table for all MTCH2 constructs in IB vs carrier treated control.