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The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup-Proteasome system (PPS). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATPase-mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity; hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions.

The N-end rule is a conserved protein degradation pathway that relates the metabolic stability of a protein to the identity of its N-terminal residue. Proteins bearing a destabilising N-terminal residue (N-degron) are recognised by specialised components of the pathway (N-recognins) and degraded by cellular proteases. In bacteria, the N-recognin ClpS is responsible for the specific recognition of proteins bearing an N-terminal destabilising residue such as leucine, phenylalanine, tyrosine or tryptophan. In this study, we show that the putative apicoplast N-recognin from Plasmodium falciparum (PfClpS), in contrast to its bacterial homologues, exhibits an expanded substrate specificity that includes recognition of the branched chain amino acid isoleucine.