FIP200 claw domain binding to p62 promotes autophagosome formation at ubiquitin condensates

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

Phasing out the bad-How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes

How RB1CC1/FIP200 claws its way to autophagic engulfment of SQSTM1/p62-ubiquitin condensates

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