A conserved loop sequence of the proteasome system depupylase Dop regulates substrate selectivity in Mycobacterium tuberculosis.

Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera. To understand the function of this domain, we deleted discrete sequences from the Dop loop and assessed pupylation in mutant strains of Mycobacterium tuberculosis. We determined that various Dop loop mutations resulted in altered pupylome profiles, in particular when mutant dop alleles were overexpressed. Taken together, our data suggest these conserved amino acids play a role in substrate selectivity for Dop.