Abstract
The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor Nub1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system and revealed that Nub1 utilizes FAT10's intrinsic instability to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin- and p97-independent manner. Through hydrogen-deuterium exchange, structural modeling, and site-directed mutagenesis, we identified the formation of a peculiar complex with FAT10 that activates Nub1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic Nub1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These studies thus identified a novel mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.