Pupylation is a post-translational modification of proteins present in the bacterial phylum Actinobacteria. The small protein Pup is bound to the target protein by a isopeptide bond and represents a mark that directs proteins to proteasomal degradation. Although pupylation is functionally analogous to ubiquitination in eukaryotes, the processes differ in the enzymes that carry them out. The reaction of pupylation is catalyzed by enzymes from the family of carboxylate amine ligases, Dop and PafA, that are also involved in depupylation and transpupylation reactions. The Pup-proteasome system in actinobacteria allows degradation of nonfunctional or damaged proteins and the activators are required to enable the entry, unfolding and translocation of substrates in the 20S core particle of the proteasome. Proteins Mpa/ARC use ATP for the proteasome activation whereas protein PafE is an activator independent of ATP. Proteasomal degradation of proteins is involved in functioning and regulation of numeorous cell processes like DNA repair, regulation of metal ions concentration in the cell and normal nitrogen metabolism. The Pup-proteasome system is necessary for pathogenicity of M. tuberculosis that causes tuberculosis, which suggests that the process of pupylation and 20S proteasome could be targets in the battle against this bacterium