The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin Andreas Küberla, Tino Polena,1, and Michael Botta,1 aIBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, 52425 Juelich, Germany Edited by Gisela Storz, National Institutes of Health, Bethesda, MD, and approved March 11, 2016 (received for review July 23, 2015) The balance of sufficient iron supply and avoidance of iron toxicity broad spectrum of functional categories, which might be explained by iron homeostasis is a prerequisite for cellular metabolism and by a general recycling function fulfilled by pupylation. In this view, growth. Here we provide evidence that, in Actinobacteria, pupyla- protein degradation mediated by pupylation is assumed to recycle tion plays a crucial role in this process. Pupylation is a posttransla- amino acids under several stress conditions in Mycobacterium tional modification in which the prokaryotic ubiquitin-like protein smegmatis (20). Although pupylation was shown to target proteins Pup is covalently attached to a lysine residue in target proteins, thus to proteasome-mediated degradation, not all pupylated proteins resembling ubiquitination in eukaryotes. Pupylated proteins are are subject to this fate (15, 21). Furthermore, the activity of the recognized and unfolded by a dedicated AAA+ ATPase (Mycobacte- mycobacterial ATPase Mpa itself was shown to be reversibly reg- rium proteasomal AAA+ ATPase; ATPase forming ring-shaped com- ulated by pupylation, which renders Mpa functionally inactive (22). plexes). In Mycobacteria, degradation of pupylated proteins by the In view of these results, the investigation of pupylation in protea- proteasome serves as a protection mechanism against several stress some-lacking Actinobacteria promises new insights into its physi- conditions. Other bacterial genera capable of pupylation such as Co- ological role(s) and the fate of pupylated proteins. rynebacterium lack a proteasome, and the fate of pupylated proteins Corynebacterium glutamicum is a member of the Actinobacteria is unknown. We discovered that Corynebacterium glutamicum mu- harboring genes for the pupylation machinery (pup, dop, pafA, tants lacking components of the pupylation machinery show a arc;Fig.1A) but lacking the proteasomal genes prcAB. In a recent strong growth defect under iron limitation, which was caused by proteomics study, we identified 55 pupylated proteins in this the absence of pupylation and unfolding of the iron storage protein species (23). C. glutamicum is a nonpathogenic Gram-positive soil ferritin. Genetic and biochemical data support a model in which the bacterium, which has become a model organism for studying pupylation machinery is responsible for iron release from ferritin in- metabolism and regulation (24). As the physiological function of dependent of degradation. pupylation in this organism remained enigmatic, we screened for a phenotype of the Δpup mutant during growth with various iron limitation | prokaryotic ubiquitin-like protein | ATPase ARC | Corynebacterium | Mycobacterium carbon sources and under different stress conditions. A severe growth defect was observed only under iron limitation. Detailed studies revealed that this phenotype is caused almost exclusively by upylation is a posttranslational protein modification occur- the lack of pupylation of the iron-storage protein ferritin, and we Pring in the phylum Actinobacteria and some other bacterial provide evidence that this could result from a defect in iron release lineages, such as Nitrospirae (1, 2). It resembles eukaryotic from nonpupylated ferritin. These results disclose a distinctive role ubiquitination and was first identified in Mycobacterium tuber- culosis (3). Target proteins are covalently linked to the small prokaryotic ubiquitin-like protein (Pup), which, in mycobacteria, Significance can serve as a tag for degradation via the proteasome (3–5). The proteasomal genes prcA and prcB are encoded within the Pupylation is a posttranslational protein modification discovered pup gene cluster of mycobacteria (6). However, several Actino- in Mycobacterium tuberculosis in which it tags proteins for bacteria harbor genes of the pupylation machinery but lack the degradation via the proteasome. It thus resembles eukaryotic genes encoding the proteasome, raising the question of the fate ubiquitination. In mycobacteria, pupylation plays a role under of pupylated proteins in proteasome-free species (1, 6). oxidative stress and under carbon and nitrogen starvation. In- The process of Pup-mediated protein degradation in myco- triguingly, many bacteria containing the pupylation machinery bacteria comprises several steps. First, the 64-aa residue-protein lack a proteasome, such as corynebacteria, leaving the function Pup is activated via deamidation of the C-terminal glutamine of this protein modification open. We show that pupylation in residue to glutamate, catalyzed by the deamidase of Pup (Dop) Corynebacterium glutamicum plays a dedicated role in iron ho- (3,7,8).Pupisthencovalentlyattachedtotargetproteinsbythe meostasis by targeting the iron-storage protein ferritin. Evidence proteasome accessory factor A (PafA). PafA catalyzes the ATP- is provided that pupylation triggers the disassembly of 24-meric dependent formation of an isopeptide bond between the γ-carboxyl ferritin by ARC to support the release of the stored iron without group of Pup and the e-amino group of a lysine residue within the using a protease. Thus, a physiological function of pupylation target protein (8–10). Mycobacterium proteasomal AAA+ ATPase was discovered for a proteasome-free bacterial species. (Mpa), termed ATPase forming ring-shaped complexes (ARCs) in Author contributions: A.K., T.P., and M.B. designed research; A.K. performed research; A.K., nonmycobacterial species, recognizes pupylated proteins, unfolds T.P., and M.B. analyzed data; and A.K., T.P., and M.B. wrote the paper. them, and directs them into the proteasome for degradation (11, The authors declare no conflict of interest. 12). Besides its function as a deamidase, Dop was also shown to This article is a PNAS Direct Submission. catalyze the depupylation of substrates (13, 14). Some species, such as members of the genera Corynebacterium and Streptomyces,en- Freely available online through the PNAS open access option. code Pup variants with a carboxyl-terminal glutamate residue, Data deposition: The DNA microarray data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. which therefore do not require the deamidation step. In these GSE64866). bacteria, Dop may serve exclusively as depupylase. 1To whom correspondence may be addressed. Email: [email protected] or m.bott@ Hitherto, proteome-wide searches revealed several pupylated fz-juelich.de. proteins (pupylomes) in a range of proteasome-bearing Actino- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bacteria (15–19). The proteins making up the pupylomes covered a 1073/pnas.1514529113/-/DCSupplemental. 4806–4811 | PNAS | April 26, 2016 | vol. 113 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1514529113 Downloaded by guest on September 29, 2021 ARC and Dop Are Also Involved in the Adaptation to Iron Limitation. A pafA pup dop arc In M. tuberculosis, pupylated proteins are recognized by the cg1688 cg1689 cg1690 cg1691 AAA+ ATPase Mpa, which unfolds Pup targets for subsequent ■-WT ■-WT ■-WT proteasomal degradation. We therefore tested whether ARC, BCDΔ - pup pup Δ - arc arc - dop dop Δ Δ ◊ Δ (-) the homolog of Mpa in C. glutamicum, is also required for op- ■-Δpup ■-Δarc ●-Δdop pup ○-Δpup pupE64A ■-Δdop timal growth under iron limitation. A mutant with an in-frame - dop dop Δ Δ (+) deletion of arc was constructed and exhibited a growth defect under iron limitation similar to the Δpup mutant, which could 10 be reversed by plasmid-based expression of arc using plasmid pVWEx1-arc (Fig. 1C). 600nm Besides Pup and ARC, the deamidase/depupylase Dop plays OD an important role during pupylation. An in-frame dop deletion 1 mutant of C. glutamicum also showed a growth defect under iron 010200 10 20 0 10 20 limitation, which was, however, not as severe as the one observed for the Δpup and Δarc mutants. The Δdop mutant reached ap- proximately 50% of the final biomass of the WT (Fig. 1D), in- Fig. 1. Importance of the pupylation machinery for growth of C. glutamicum dicating that Dop is required for optimal adaptation to iron ATCC 13032 under iron limitation. (A) Genetic organization of pupylation- limitation, but not as important as Pup or ARC. In contrast to related genes. (B–D) Growth of the WT (black squares) and pupylation- the successful complementation of the Δpup and Δarc mutants, deficient mutants cultivated in glucose minimal medium under iron limita- IPTG-induced overexpression of the dop gene in the Δdop strain tion (1 μM FeSO4). The strains are indicated at the top and represent: (B) Δpup (red squares), Δpup/pVWEx1-pup (red triangles), and Δpup/pVWEx1- with plasmid pVWEx1-dop resulted in an even more pronounced pupE64A (open circles); (C) Δarc/pVWEx1 (green squares) and Δarc/pVWEx1- growth defect under iron limitation rather than in its suppression arc (green triangles); and (D) Δdop/pVWEx1 (blue squares) and Δdop/ (Fig. 1D). In contrast, no growth defect caused by dop over- pVWEx1-dop in the presence (blue triangles) and absence (blue diamonds) expression was observed