Proteomic screening of TMEM43 binding partners identifies VDAC leading to mitochondrial dysfunction

Figures

• 

  Fig 1. IP-MS systematically determined the differential binding differential binding proteins of TMEM43 versus TMEM43 p.S358L mutant. (A) The flow chart of IP-MS assay. (B) Principal Component Analysis (PCA) of MS result showed the three clusters of the IP samples, suggesting the significant differentiation between the three groups. T43 TMEM43; Mu Mutant; Vc Vector (C) Venn diagram showed 112 and 40 proteins were found to bind to TMEM43 and TMEM43 p.S358L mutant respectively, and another 126 proteins were seen to bind to both of them. (D) The volcano plot of DBPs in TMEM43 versus TMEM43 p.S358L mutant. Important features were determined by volcano plot with fold change (FC) threshold and t-tests threshold. The blue dots and red dots represent the proteins of significantly decreasing binding and increasing binding ability of TMEM43, respectively.

• 

  Fig 2. The pathway enrichments of differential binding proteins of TMEM43 between cells expressing TMEM43 and TMEM43 p.S358L mutant. (A) Scatterplot of enriched KEGG pathways of DBPs in TMEM43 versus TMEM43 p.S358L mutant. (B) Scatterplot of enriched diseases of DBPs in TMEM43 versus TMEM43 p.S358L mutant. Both enrichments highlight arrhythmogenic right ventricular cardiomyopathy (ARVC) and cardiovascular diseases. The permission of KEGG usage in this article was kindly provided by Kanehisa Laboratories [20].

• 

  Fig 3. The interaction of TMEM43 and its S358L mutant with several selected binding partners were confirmed in A549 cells. (A) The Immunofluorescence (IF) staining showed the partially colocalization of HA-Flag-TMEM43 and its S358L mutant with the selected binding partners including γActin (ACTG1), Caveolin 1 (CAV1), and APOB in A549 cells. A549 cells stably expressing HA-Flag-TMEM43 and its S358L mutant were stained with DAPI, HA antibody or Flag antibody, and the indicated antibodies. The pictures were taken under fluorescent microscope. scale bar: 10um (B) The colocalization percentage in 7 random regions of IF staining under 200 magnifications was counted and compared. Student t-test was performed. * indicates p < 0.05, ** indicates p < 0.01.

• 

  Fig 4. S358L mutation reduced the interaction of TMEM43 with VDACs. (A) IF staining showed the colocalization of Flag-TMEM43 and its S358L mutant (green) with VDAC1 and VDAC2 (red) in cardiac myoblast H9c2 cells. H9c2 cells stably expressing HA-Flag-TMEM43 and its S358L mutant were stained with DAPI, and the indicated antibodies. Scale bar: 10um (B) The colocalization percentage in 6 random regions of IF staining under 200 magnifications was counted and compared. (C) Immunoblotting with indicated antibodies displayed the protein expression of VDAC2, VDAC1 and Flag-TMEM43 before and after Flag IP. (D) The lysate of mouse hearts was IP with indicated antibodies and submitted to immunoblotting.

• 

  Fig 5. Mitochondrial dysfunction in the TMEM43p.S358L H9c2 cells. (A) MitoTracker red staining indicated the mitochondrial morphology in H9c2 cells stably expressing HA-Flag-TMEM43 and its S358L mutant. H9c2 cells stably expressing HA-Flag-TMEM43 and its S358L mutant were stained with DAPI, and MitoTracker red. Magnification 100X, Scale bar: 4um (B) Flow cytometry exhibited the cell percentage of JC-1 monomer (FITC) and aggregate (PE). Quantification of mitochondrial membrane potential (the ratio of JC-1 aggregate/ monomer) was performed with triple analyses of flow cytometry. (C) The Ca2 + level and mitochondrial ROS level in TMEM43 and mutant H9c2 cells. Fluo-4 AM fluorescent staining (top panel) indicated the intracellular Ca2 + level in H9c2 cells stably expressing HA-Flag-TMEM43 and its S358L mutant. MitoSOX fluorescent staining (bottom panel) indicated the mitochondrial ROS level in H9c2 cells stably expressing HA-Flag-TMEM43 and its S358L mutant. Magnification 100X, Scale bar: 100um. Quantification of fluorescence intensity was performed for both assays.