The prokaryotic ubiquitin-like protein presents poor cleavage sites for proteasomal degradation.

In an event reminiscent of eukaryotic ubiquitination, the bacterial prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS) marks target proteins for proteasomal degradation by covalently attaching Pup, the bacterial tagging molecule. Yet, ubiquitin is released from its conjugated target following proteasome binding, whereas Pup enters the proteasome and remains conjugated to the target. Here, we report that although Pup can be degraded by the bacterial proteasome, it lacks favorable 20S core particle (CP) cleavage sites and is thus a very poor 20S CP substrate. Reconstituting the PPS in vitro, we demonstrate that during pupylated protein degradation, Pup can escape unharmed and remain conjugated to a target-derived degradation fragment. Removal of this degradation fragment by Dop, a depupylase, facilitates Pup recycling and re-conjugation to a new target. This study thus offers a mechanistic model for Pup recycling and demonstrates how a lack of protein susceptibility to proteasome-mediated cleavage can play a mechanistic role in a biological system.