RESUMO
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.
Assuntos
Mitofagia , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Autofagossomos/metabolismo , Proteínas Reguladoras de Apoptose , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , AutofagiaRESUMO
The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.
Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Proteínas Relacionadas à Autofagia , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genéticaRESUMO
Macroautophagy/autophagy mediates the degradation of ubiquitinated aggregated proteins within lysosomes in a process known as aggrephagy. The cargo receptor SQSTM1/p62 condenses aggregated proteins into larger structures and links them to the nascent autophagosomal membrane (phagophore). How the condensation reaction and autophagosome formation are coupled is unclear. We recently discovered that a region of SQSTM1 containing its LIR motif directly interacts with RB1CC1/FIP200, a protein acting at early stages of autophagosome formation. Determination of the structure of the C-terminal region of RB1CC1 revealed a claw-shaped domain. Using a structure-function approach, we show that the interaction of SQSTM1 with the RB1CC1 claw domain is crucial for the productive recruitment of the autophagy machinery to ubiquitin-positive condensates and their subsequent degradation by autophagy. We also found that concentrated Atg8-family proteins on the phagophore displace RB1CC1 from SQSTM1, suggesting an intrinsic directionality in the process of autophagosome formation. Ultimately, our study reveals how the interplay of SQSTM1 and RB1CC1 couples cargo condensation to autophagosome formation.