Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor

Abstract

Pupylation is a posttranslational modification peculiar to actinobacteria wherein proteins are covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated protein degradation in mycobacteria. Here, we report studies of pupylation in a streptomycete that is phylogentically related to mycobacteria. We constructed mutants of Streptomyces coelicolor lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these mutants share a high susceptibility to oxidative stress compared to that of the wild-type strain. Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking genes encoding the 20S proteasome. Our findings shed new light on this unusual posttranslational modification and its role in Streptomyces physiology. IMPORTANCEThe presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have motivated reevaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome system has been studied extensively in mycobacteria, it is much less understood in streptomycetes, members of a large genus of actinobacteria known for highly choreographed life cycles in which phases of morphological differentiation, sporulation, and secondary metabolism are often regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces coelicolor for the first time and present new evidence that links pupylation and the oxidative stress response in this bacterium. Surprisingly, we found that the Pup ligase has a Pup-independent role in sporulation.

The Absence of Pupylation (Prokaryotic Ubiquitin-Like Protein Modification) Affects Morphological and Physiological Differentiation in Streptomyces coelicolor

“…Our results are consistent with those of Compton et al, who found that a S. coelicolor pafA-null strain displayed defects of both sporulation and secondary metabolism and who also presented evidence linking pupylation and the oxidative stress response in S. coelicolor (60). The differences in the phenotypes of the pup and proteasome mutants observed in our study indicate that pupylation may have other roles in addition to its known proteasome-associated function.…”

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

“…In mycobacteria and streptomycetes, pupylation was shown to target proteins to proteasomal degradation and to play an important role in the response to several stress conditions, such as nitrosative stress (25), oxidative stress (19), or nitrogen and carbon starvation (20), as well as in the development of cell morphology (21). However, members of other actinobacterial genera such as Corynebacterium do not harbor a prcAB-encoded proteasome; nevertheless, they possess the genes for the pupylation machinery including Pup, Dop, PafA, and Mpa/ARC.…”

“…Hitherto, proteome-wide searches revealed several pupylated proteins (pupylomes) in a range of proteasome-bearing Actinobacteria (15)(16)(17)(18)(19). The proteins making up the pupylomes covered a broad spectrum of functional categories, which might be explained by a general recycling function fulfilled by pupylation.…”

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

“…In Streptomyces coelicolor, disruption of pupylation was recently shown to cause a sporulation defect [48].…”

“…Irrespective of the roles played by individual degradation substrates or groups of substrates, it is well-supported that the substrate clientele consisting of pupylated proteins (the so-called pupylome) for the proteasome complex under certain growth conditions encompasses a hundred or more cellular proteins differing in structure, assembly state and abundance [48][49][50][51]. Their unifying recognition property is the covalent modification of one or more of their lysine side chains with Pup, which mediates their interaction with a ring-shaped, hexameric proteasomal ATPase complex referred to as Mpa in mycobacteria or ARC in other actinobacteria.…”