Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.