TGFB signaling induces mitophagy via PLSCR3-mediated cardiolipin externalization in conjunction with a BNIP3L/NIX-, BNIP3-, and FUNDC1-dependent mechanism

Selective clearance of damaged mitochondria through mitophagy is crucial for the maintenance of mitochondrial homeostasis. While mitophagy can be activated by various mitochondrial toxins, the physiologically relevant signal that triggers mitophagy is less studied. TGFB/TGFβ signaling has been linked to autophagic induction, but its specific role in mitophagy is not well understood. Here, we discovered a novel mitophagy induction paradigm stimulated by TGFB1. The mitophagic response is exclusively mediated by SMAD2, SMAD3, and SMAD4 underlying the TGFB receptor signaling. The transcriptional regulation activates genes involved in the canonical autophagic pathway which is required for the TGFB1-induced mitophagy. Moreover, TGFB1 signaling promotes mitophagic flux by upregulating PLSCR3 that externalizes cardiolipin in conjunction with the MAP1LC3/LC3/GABARAPs-interacting receptor proteins (BNIP3L/NIX, BNIP3, and FUNDC1)-dependent mechanism. Overall, our study characterized the essential components engaged in the TGFB1-induced mitophagy and demonstrated that TGFB is an important signal that induces mitophagy. <b>Abbreviations</b> ATG5: autophagy related 5; ATG8: mammalian homolog of yeast Atg8; ATG9A: autophagy related 9A; ATG13: autophagy related 13; ATG101: autophagy related 101; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Cardiolipin: 1,3-bis(<i>sn</i>-3’-phosphatidyl)-<i>sn</i>-glycerol; CERS1: ceramide synthase 1; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GLS: glutaminase; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MitoIP: mitochondrial immunoprecipitation; MMP: mitochondrial membrane potential; NRBF2: nuclear receptor binding factor 2; OPTN: optineurin; PINK1: PTEN induced kinase 1; PLSCR3: phospholipid scramblase 3; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TGFB/TGFβ: transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1.