Unconventional initiation of PINK1/Parkin mitophagy by Optineurin.

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 or 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.

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.

Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin (PINK1/Parkin)-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 (TANK-binding kinase 1) adaptors NAP1 (NAK-associated protein 1) and SINTBAD (similar to NAP1 TBK1 adaptor) restrict the initiation of OPTN (optineurin)-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of nuclear dot protein 52 (NDP52)-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.

ATG4s: above and beyond the Atg8-family protein lipidation system.

The sole proteases of the macroautophagy/autophagy machinery, the ATG4s, contribute to autophagosome formation by cleaving Atg8-family protein members (LC3/GABARAPs) which enables Atg8-family protein lipidation and de-lipidation. Our recent work reveals that ATG4s can also promote phagophore growth independently of their protease activity and of Atg8-family proteins. ATG4s and their proximity partners including ARFIP2 and LRBA function to promote trafficking of ATG9A to mitochondria during PINK1-PRKN mitophagy. Through the development of a 3D electron microscopy framework utilizing FIB-SEM and artificial intelligence (termed AIVE: Artificial Intelligence-directed Voxel Extraction), we show that ATG4s promote ER-phagophore contacts during the lipid-transfer phase of autophagosome biogenesis, which requires ATG2B and ATG9A to support phagophore growth. We also discovered that ATG4s are not essential for removal of Atg8-family proteins from autolysosomes, but they can function as deubiquitinase-like enzymes to counteract the conjugation of Atg8-family proteins to other proteins, a process that we have termed ATG8ylation (also known as LC3ylation). These discoveries demonstrate the duality of the ATG4 family in driving autophagosome formation by functioning as both autophagy proteases and trafficking factors, while simultaneously raising questions about the putative roles of ATG8ylation in cell biology.

LC3/GABARAPs drive ubiquitin-independent recruitment of Optineurin and NDP52 to amplify mitophagy.

Current models of selective autophagy dictate that autophagy receptors, including Optineurin and NDP52, link cargo to autophagosomal membranes. This is thought to occur via autophagy receptor binding to Atg8 homologs (LC3/GABARAPs) through an LC3 interacting region (LIR). The LIR motif within autophagy receptors is therefore widely recognised as being essential for selective sequestration of cargo. Here we show that the LIR motif within OPTN and NDP52 is dispensable for Atg8 recruitment and selectivity during PINK1/Parkin mitophagy. Instead, Atg8s play a critical role in mediating ubiquitin-independent recruitment of OPTN and NDP52 to growing phagophore membranes via the LIR motif. The additional recruitment of OPTN and NDP52 amplifies mitophagy through an Atg8-dependent positive feedback loop. Rather than functioning in selectivity, our discovery of a role for the LIR motif in mitophagy amplification points toward a general mechanism by which Atg8s can recruit autophagy factors to drive autophagosome growth and amplify selective autophagy.

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs.

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.

Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.

Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome-lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome-lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.