A kinetic model for the prevalence of mono‐ over poly‐pupylation

Abstract

Bacteria belonging to the phyla Actinobacteria and Nitrospira possess proteasome cores homologous to the eukaryotic 20S proteasome particle. In these bacteria, the cytoplasmic signal for proteasomal degradation is a small protein termed Pup (prokaryotic ubiquitin‐like protein). PafA, the only known Pup ligase, conjugates Pup to lysine side chains of target proteins. In contrast to the eukaryotic ubiquitin‐proteasome system, where poly‐ubiquitin chains are the principal tags for proteasomal degradation, mono‐Pup moieties are almost exclusively observed in vivo and are sufficient as degradation tags. Although Pup presents lysines, raising the possibility of poly‐Pup chain assembly, these do not predominate. At present, the factors promoting the distinct predominance of mono‐ over poly‐pupylation remain poorly understood. To address this issue, we conducted a detailed biochemical analysis characterizing the pupylation of model proteins in vitro. We found that Pup can indeed serve as a pupylation target for PafA either in its free form or when already conjugated to proteins, thus allowing for the formation of poly‐Pup chains. However, our results indicate that pupylation of an already pupylated protein is unlikely to occur due to low affinity of PafA for such species. This alone prevents predominance of poly‐ over mono‐pupylation in vitro. This effect is likely to be magnified in vivo by the combination of PafA kinetics with the high abundance of non‐pupylated proteins. Overall, this work provides a kinetic explanation for the prevalence of mono‐ rather than poly‐pupylation in vivo, and sheds light on PafA substrate specificity.

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

“…This notion is supported by the fact that many M. smegmatis and M. tuberculosis HMW proteins were not detected in the pupylomes of these species (5,9,15,16). It is further supported by the fact that a good substrate can be converted into a poor substrate by mutagenesis of its target lysine (10,27), a mutation that hardly affects its size and the accessibility of other lysines in the protein.…”

“…All proteins used in this study were recombinant M. smegmatis proteins, with the exception of Ac-Dop. PafA, Pup, and PanB were purified as described by Ofer et al (34), Dop variants and Pup-Fl were purified as described by Hecht and Gur (24), and IdeR, pupylated PanB, and pupylated IdeR were purified as described by Regev et al (10). The M. smegmatis pupylome was purified as described in Elharar et al (5).…”

“…Following depupylation, the 5-FAM-lysine moiety is released from Pup, as can be easily detected by SDS/PAGE followed by fluorescence scanning of the gel (24). The third and fourth substrates were pupylated PanB and IdeR, two bona fide M. smegmatis pupylation targets (10). PanB is a homo-decamer comprising 31-kDa subunits, whereas IdeR is a homo-dimer of 26-kDa subunits.…”

“…Pup is encoded by the first gene of the prcSBA operon, with the remaining genes, prcB and prcA, encoding the proteasome 20S core particle (20S CP) β and α subunits, respectively. Unlike ubiquitin, Pup is natively unfolded (7,8), and poly-Pup chains are rarely assembled (9,10). The Pup moiety of a modified protein is recognized by the proteasome regulatory particle termed Mpa in mycobacteria and Arc in nonmycobacterial species (11)(12)(13).…”

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

“…An in vitro study showed for two known pupylation substrates (IdeR and PanB) that poly-pupylation, when it occurs, proceeds via sequential addition of Pup to a growing Pup chain on the substrate rather than by attaching an existing poly-Pup [76]. The study further concludes that in case the affinity for mono-pupylated substrate is similar to unpupylated target as for IdeR, polypupylation occurs along with monopupylation, whereas for much higher affinity of unpupylated target, as is the case for PanB, monopupylation is preferred.…”