Abstract
This chapter describes the identification of the first prokaryotic ubiquitin-like protein modifier, Pup, which covalently attaches to proteins to target them for destruction by a bacterial proteasome in a manner akin to ubiquitin in eukaryotes. Despite using a proteasome as the end point for proteolysis, Pup and ubiquitin differ in sequence, structure and method of activation and conjugation to protein substrates. Pup is so far the only known posttranslational protein modifier in prokaryotes and its discovery opens the door to the possibility that others are present not only for proteolysis, but also to regulate protein function or localization. Here, we discuss the putative mechanism of activation and conjugation of Pup (termed "pupylation") to target proteins. In addition, because it is unclear whether or not Pup, like ubiquitin, is recycled or degraded during substrate targeting to the proteasome, we propose methods that may identify Pup deconjugation enzymes ("depupylases"). Finally, we outline future directions for Pup research and anti-tuberculosis drug discovery.