Autophagy
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The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an in vivo system in which mitophagy is slowed by inhibiting mitochondrial division through knockout of Dnm1l/Drp1, a dynamin related GTPase that mediates mitochondrial division. Using this system, we revealed that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/parkin, during mitophagy. ⋯ These data suggest that mitochondrial ubiquitination is promoted by SQSTM1 independently of PINK1 and PRKN during mitophagy. PINK1 and PRKN appear to control the balance between mitochondrial division and fusion in vivo. Abbreviations: DNM1L/DRP1: dynamin 1-like; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN1/2: mitofusin 1/2; OPA1: OPA1, mitochondrial dynamin like GTPase; PDH: pyruvate dehydrogenase E1; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase.
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Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from in vitro experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. ⋯ As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel. Abbreviations: BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco's Modified Eagle's Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks' balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ΔΨm: mitochondrial membrane potential.