Cells were further treated with CCCP seeing that indicated. to explore the role of MIEF1 in apoptosis. MIEF1 loss brought on the imbalance of BCL2 family members around the mitochondria, consequently initiating the translocation of BAX onto the mitochondria, catalyzing the decrease of mitochondrial membrane potential and promoting the release of DIABLO/SMAC (diablo IAP-binding mitochondrial protein) and CYCS (cytochrome c, somatic). We further demonstrate that MIEF1 deficiency impaired mitochondrial respiration and induced mitochondrial oxidative stress, sensitizing cells to PINK1-PRKN-mediated mitophagy. The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate. Abbreviations: ActD: actinomycin D; BAX: BCL2 associated X, apoptosis regulator; BAK1: BCL2 antagonist/killer 1; BCL2L1: BCL2 like 1; BMH: 1,6-bismaleimidohexane; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHX: cycloheximide; CQ: o-Cresol chloroquine; CYCS: cytochrome c, somatic; DIABLO: diablo IAP-binding mitochondrial protein; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; FIS1: fission, mitochondrial 1; GFP: green fluorescent protein; IP: immunoprecipitation; MFN1: mitofusin 1; MFN2: mitofusin 2; MG132: carbobenzoxy-Leu-Leu-leucinal; MIEF1/MiD51: mitochondrial elongation factor 1; MIEF2/MiD49: mitochondrial elongation factor 2; MOMP: mitochondrial outer membrane permeabilization; MTR: MitoTracker Red; OA: oligomycin plus antimycin A; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; SD: standard deviation; STS: staurosporine; TNF: tumor o-Cresol necrosis o-Cresol factor; UPS: ubiquitin-proteasome system; VDAC1: voltage dependent anion channel o-Cresol 1. fails to interfere with apoptosis programming [27C30], bringing into question that whether mitochondrial fission is usually preliminary for apoptosis. Thus, more characterization of mitochondrial dynamics proteins in the regulation of cell death requires to be studied. Mitochondrial-associated apoptosis results in gross production of reactive oxygen species (ROS) inevitably [31]. However, in addition to apoptotic cell death sentences, cells can also utilize an alternative pathway to remove aberrant mitochondria, which is usually mediated by the selective autophagy, known as mitophagy [32C34]. The most theory mitophagic pathway is the PINK1-PRKN-dependent route. Upon loss of mitochondrial membrane potential, the PINK1 (PTEN induced kinase 1) stabilizes on OMM [35C37], phosphorylating ubiquitin and PRKN (parkin RBR E3 ubiquitin protein ligase), which promotes the E3 ligase activity of PRKN, leading to further deposition of ubiquitin and PRKN accumulation onto the mitochondria [38,39]. PRKN subsequently mediates the ubiquitination and degradation of mitochondrial resident proteins, including MFN1 (mitofusin 1), MFN2 and VDAC1 (voltage dependent anion channel 1) via the ubiquitin-proteasome pathway [40C43]. This feed-forward mechanism essentially triggers the engulfment of mitochondria by ubiquitin adaptors, resulting in mitochondrial clearance through lysosomal degradation [44C46]. Physiologically, PINK1-PRKN-mediated mitophagy is usually Mouse monoclonal to PTK7 highly pronounced in pathogenicity of neuronal diseases, particularly Parkinson disease [47,48]. Mutations of PINK1 and PRKN have been found in Parkinson disease, suggesting the underlying physiological importance of PINK1-PRKN-dependent mitophagy. It is intensively studied that in cultured cells, acute mitochondrial damage and toxification are required to induce the PINK1-PRKN pathway. The mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is usually widely used to depolarize mitochondria, triggering the translocation of PRKN onto damaged mitochondria. However, very little is known about the threshold level of vulnerability of mitochondria to toxins, which may primary cells to mitophagy. MIEF1 is an outer mitochondrial membrane protein, made up of a single-pass transmembrane domain name at the N-terminus, which anchors the protein to the mitochondria, with the bulk of the protein facing the cytosol. MIEF1 was simultaneously identified with MIEF2, which similarly mediates the mitochondrial fission machinery via DNM1L [49,50]. Overexpression of MIEF1 or o-Cresol MIEF2 sequesters excessive inactive DNM1L on OMM,.