Unconventional Initiation of PINK1/Parkin Mitophagy by Optineurin

Abstract

Cargo sequestration is a fundamental step of selective autophagy in which cells generate a double membrane structure termed an autophagosome on the surface of cargoes. NDP52, TAX1BP1 and p62 bind FIP200 which recruits the ULK1/2 complex to initiate autophagosome formation on cargoes. How OPTN initiates autophagosome formation during selective autophagy remains unknown despite its importance in neurodegeneration. Here, we uncover an unconventional path of PINK1/Parkin mitophagy initiation by OPTN that does not begin with FIP200 binding nor require the ULK1/2 kinases. Using gene-edited cell lines and in vitro reconstitutions, we show that OPTN utilizes the kinase TBK1 which binds directly to the class III phosphatidylinositol 3-kinase complex I to initiate mitophagy. During NDP52 mitophagy initiation, TBK1 is functionally redundant with ULK1/2, classifying TBK1’s role as a selective autophagy initiating kinase. Overall, this work reveals that OPTN mitophagy initiation is mechanistically distinct and highlights the mechanistic plasticity of selective autophagy pathways.

Structure and activation of the human autophagy-initiating ULK1C:PI3KC3-C1 supercomplex

“…The higher resolution analysis here confirms the previous observation that the ULD and the Arm domain of FIP200 have the same fold as the scaffold domain of the Tank-binding kinase 1 (TBK1) 20 (Extended Data Fig. 2b), which is itself central to the initiation of some forms of autophagy 28 .…”

“…2b and Extended Data Fig. 7h), and TBK1 has been shown to bind directly to PI3KC3-C1 28 . It will be interesting to determine if TBK1 uses its FIP200 arm-like domain to bypass the need for ULK1/2 in OPTN mitophagy.…”

“…We therefore tested the function of both single and multisite FIP200 mutations. The first steps in PINK1/Parkin mitophagy can bypass ULK1 recruitment via the OPTN-TBK1-PI3KC3-C1 axis, while NDP52 can utilize either ULK1 or TBK1, 31 and therefore a full loss of function was not expected. Most FIP200 mutations did not lead to an evident loss of function.…”

“…Mitophagy induction was carried out as described in refs. 31,54 . Briefly, 350,000 cells were seeded the day before the mitophagy induction in 6 well plates.…”

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery

“…Phosphorylation of autophagy adaptors near their LIR motifs by TBK1 enhances the adaptors’ ability to interact with and recruit mAtg8 proteins and/or the ULK1/FIP200 complex (Vargas et al, 2019; Wild et al, 2011). TBK1 can also promote mitophagy through phosphorylation of RAB7A (Heo et al, 2018) or by directly activating the phosphatidylinositol 3-kinase (PI3K) complex I (Nguyen et al, 2023). By activating TBK1, TRIM5α is upstream of all of these processes.…”