TBK1 adaptor AZI2/NAP1 regulates NDP52-driven mitochondrial autophagy.

Damaged mitochondria are selectively eliminated in a process called mitophagy. PINK1 and Parkin amplify ubiquitin signals on damaged mitochondria, which are then recognized by autophagy adaptors to induce local autophagosome formation. NDP52 and OPTN, two essential mitophagy adaptors, facilitate de novo synthesis of pre-autophagosomal membranes near damaged mitochondria by linking ubiquitinated mitochondria and ATG8 family proteins and by recruiting core autophagy initiation components. The multifunctional serine/threonine kinase TBK1 also plays important roles in mitophagy. OPTN directly binds TBK1 to form a positive feedback loop for isolation membrane expansion. TBK1 is also thought to indirectly interact with NDP52; however, its role in NDP52-driven mitophagy remains largely unknown. Here, we focused on two TBK1 adaptors, AZI2/NAP1 and TBKBP1/SINTBAD, that are thought to mediate the TBK1-NDP52 interaction. We found that both AZI2 and TBKBP1 are recruited to damaged mitochondria during Parkin-mediated mitophagy. Further, a series of AZI2 and TBKBP1 knockout constructs combined with an OPTN knockout showed that AZI2, but not TBKBP1, impacts NDP52-driven mitophagy. In addition, we found that AZI2 at S318 is phosphorylated during mitophagy, the impairment of which slightly inhibits mitochondrial degradation. These results suggest that AZI2, in concert with TBK1, plays an important role in NDP52-driven mitophagy.

Optineurin provides a mitophagy contact site for TBK1 activation.

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.