p62 filaments capture and present ubiquitinated cargos for autophagy

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin‐proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62‐dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross‐linked by the substrates. The reaction is inhibited by free ubiquitin, K48‐, and K63‐linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy. Synopsis p62 and ubiquitinated substrates spontaneously coalesce into larger clusters, which result from the crosslinking of p62 filaments by the substrates. Efficient cluster formation requires substrates of at least 2 ubiquitin chains longer than 3 ubiquitin moieties. Substrate‐attached M1‐, K48 and K63‐linked ubiquitin chains all induce cluster formation. Free mono‐ubiquitin as well as free K48‐ and K63‐linked chains inhibit clustering. Addition of LC3 and mutation of the LC3‐interacting region (LIR) of p62 reduces cluster formation. Graphical Abstract In a reconstituted system, the selective autophagy cargo receptor p62 and ubiquitinated substrates spontaneously coalesce into larger clusters due to crosslinking of p62 filaments by the substrates.