Bacteria belonging to the phyla Actinobacteria and Nitrospira possess proteasome cores homologous to the eukaryotic 20S proteasome particle. In these bacteria, the cytoplasmic signal for proteasomal degradation is a small protein termed Pup (prokaryotic ubiquitin‐like protein). PafA, the only known Pup ligase, conjugates Pup to lysine side chains of target proteins. In contrast to the eukaryotic ubiquitin‐proteasome system, where poly‐ubiquitin chains are the principal tags for proteasomal degradation, mono‐Pup moieties are almost exclusively observed in vivo and are sufficient as degradation tags. Although Pup presents lysines, raising the possibility of poly‐Pup chain assembly, these do not predominate. At present, the factors promoting the distinct predominance of mono‐ over poly‐pupylation remain poorly understood. To address this issue, we conducted a detailed biochemical analysis characterizing the pupylation of model proteins in vitro. We found that Pup can indeed serve as a pupylation target for PafA either in its free form or when already conjugated to proteins, thus allowing for the formation of poly‐Pup chains. However, our results indicate that pupylation of an already pupylated protein is unlikely to occur due to low affinity of PafA for such species. This alone prevents predominance of poly‐ over mono‐pupylation in vitro. This effect is likely to be magnified in vivo by the combination of PafA kinetics with the high abundance of non‐pupylated proteins. Overall, this work provides a kinetic explanation for the prevalence of mono‐ rather than poly‐pupylation in vivo, and sheds light on PafA substrate specificity.

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Conjugation of the prokaryotic ubiquitin-like protein (Pup) to cellular proteins tags these proteins for degradation by a proteasome in actinobacteria. To study the Pup-proteasome system in in vitro biochemical assays, Pup-tagged (i.e., pupylated) proteins are often used. However, the purification of a homogeneous preparation of pupylated proteins often suffers from poor yields and limitations in terms of selecting the target protein and its site of pupylation. Here, we report on the development of a biochemical methodology we term Pup-Click for the generation of pupylated protein mimics in vitro. Pup-Click relies on a natural pupylation reaction combined with the use of a synthetic peptide and genetic code expansion via the use of unnatural amino acids and Click chemistry. In principle, this approach allows for conjugation of Pup to any selected target at potentially any desired position. Importantly, pupylated protein mimics generated by Pup-Click are recognized and processed by enzymes of the Pup-proteasome system. As such, Pup-Click can serve as a powerful tool for studying this protein degradation pathway.