Mitochondria play an essential role in maintaining cellular homeostasis. The removal of damaged or depolarized mitochondria occurs via mitophagy, in which damaged mitochondria are targeted for degradation via ubiquitination induced by PTEN-induced putative kinase 1 (PINK1) and Parkin. Mitophagy receptors, including optineurin (OPTN), nuclear dot 52 kDa protein (NDP52), and Tax1-binding protein 1 (TAX1BP1), are recruited to mitochondria via ubiquitin binding and mediate autophagic engulfment through their association with microtubule-associated protein light chain 3 (LC3). Here, we use live-cell imaging to demonstrate that OPTN, NDP52, and TAX1BP1 are recruited to mitochondria with similar kinetics following either mitochondrial depolarization or localized generation of reactive oxygen species, leading to sequestration by the autophagosome within ∼45 min after insult. Despite this corecruitment, we find that depletion of OPTN, but not NDP52, significantly slows the efficiency of sequestration. OPTN is phosphorylated by the kinase TANK-binding kinase 1 (TBK1) at serine 177; we find that TBK1 is corecruited with OPTN to depolarized mitochondria. Inhibition or depletion of TBK1, or expression of amyotrophic lateral sclerosis (ALS)-associated OPTN or TBK1 mutant blocks efficient autophagosome formation. Together, these results indicate that although there is some functional redundancy among mitophagy receptors, efficient sequestration of damaged mitochondria in response to mitochondrial stress requires both TBK1 and OPTN. Notably, ALS-linked mutations in OPTN and TBK1 can interfere with mitophagy, suggesting that inefficient turnover of damaged mitochondria may represent a key pathophysiological mechanism contributing to neurodegenerative disease.
Keywords: Parkin; TBK1; amyotrophic lateral sclerosis; mitophagy; optineurin.