Reconstitution of the <i>Mycobacterium tuberculosis</i> pupylation pathway in <i>Escherichia coli</i>

Cerda-Maira, Francisca A.; McAllister, Fiona E.; Bode, Nadine J.; Burns, Kristin E.; Gygi, Steven P.; Darwin, K. Heran

doi:10.1038/embor.2011.109

Cited by 45 publications

(59 citation statements)

References 34 publications

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“…PafA is the sole enzyme catalyzing Pup conjugation to target substrates, coupling ATP hydrolysis to the formation of an isopeptide bond between the side chains of Pup C-terminal glutamate and a substrate lysine (6,14). PafA presents a remarkable broad specificity, recognizing a variety and apparently unrelated protein targets via a mechanism that remains poorly understood (5,9,(15)(16)(17). The broad specificity of PafA correlated well with the proposed role of the PPS as an amino acid recycling factory under starvation conditions (5).…”

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confidence: 83%

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

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for highmolecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions. Dop | mycobacteria | PafA | proteasome | Pup

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confidence: 83%

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

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The migration of the pupylated protein on SDS-PAGE was altered upon pupylation of other sites on the molecule. 26) Based on the primary sequence and structural data for mycobacterial PPase, several lysine residues were exposed on the surface of Rhodococcus PPase, 27) suggesting that lysine residues other than K155 were also pupylated.…”

Section: Proteomic Analysis Of Pupylated Proteins and Preparation Of mentioning

confidence: 99%

RhodococcusProkaryotic Ubiquitin-Like Protein (Pup) Is Degraded by Deaminase of Pup (Dop)

Yun

Tamura

2012

Bioscience, Biotechnology, and Biochemistry

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“…Pupylated targets are numerous and belong to various functional classes of proteins. The mycobacterial genes pafA and pup (gene modified to encode a protein with a GGE C terminus) have been expressed in Escherichia coli, resulting in the pupylation of host proteins (31). This provides clear evidence that no mycobacterial cofactor is essential for substrate recognition by PafA.…”

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confidence: 99%

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

et al. 2015

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Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCEStreptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.T urnover of cellular proteins is required in all living organisms to maintain normal cellular physiology. In eukaryotes, the ubiquitin-proteasome pathway is the major route of protein degradation and turnover (1). Ubiquitin is a 76-residue protein that can be conjugated to target proteins, marking them for degradation by the 26S proteasome complex. In addition to its role in tagging proteins for degradation...

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Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli

Cited by 45 publications

References 34 publications

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

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

RhodococcusProkaryotic Ubiquitin-Like Protein (Pup) Is Degraded by Deaminase of Pup (Dop)

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

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