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      Real‐time detection of N‐end rule‐mediated ubiquitination via fluorescently labeled substrate probes†

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          • The N‐end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N‐end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N‐end rule E3 ligase PROTEOLYSIS1 ( PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time.

          • We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in‐gel fluorescence scanning as well as in real time by fluorescence polarization.

          • The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1‐mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption.

          • We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.

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          Most cited references 66

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          Building ubiquitin chains: E2 enzymes at work.

          The modification of proteins with ubiquitin chains can change their localization, activity and/or stability. Although ubiquitylation requires the concerted action of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s), it is the E2s that have recently emerged as key mediators of chain assembly. These enzymes are able to govern the switch from ubiquitin chain initiation to elongation, regulate the processivity of chain formation and establish the topology of assembled chains, thereby determining the consequences of ubiquitylation for the modified proteins.
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            In vivo half-life of a protein is a function of its amino-terminal residue.

            When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.
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              Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization.

              The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low-as during flooding-RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops. © 2011 Macmillan Publishers Limited. All rights reserved

                Author and article information

                New Phytol
                New Phytol
                The New Phytologist
                John Wiley and Sons Inc. (Hoboken )
                09 March 2017
                January 2018
                : 217
                : 2 ( doiID: 10.1111/nph.2018.217.issue-2 )
                : 613-624
                [ 1 ] Independent Junior Research Group on Protein Recognition and Degradation Leibniz Institute of Plant Biochemistry (IPB) Weinberg 3 Halle (Saale) D‐06120 Germany
                [ 2 ] ScienceCampus Halle – Plant‐based Bioeconomy Betty‐Heimann‐Str. 3 Halle (Saale) D‐06120 Germany
                [ 3 ] Department of Bioorganic Chemistry Leibniz Institute of Plant Biochemistry (IPB) Weinberg 3 Halle (Saale) D‐06120 Germany
                Author notes
                [* ] Author for correspondence:

                Nico Dissmeyer

                Tel: +49 345 5582 1710

                Email: nico.dissmeyer@

                © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 3, Tables: 0, Pages: 12, Words: 9987
                Funded by: ScienceCampus Halle – Plant‐based Bioeconomy
                Award ID: WE 1467/13‐1
                Funded by: German Research Foundation (DFG)
                Award ID: DI 1794/3‐1
                Funded by: Leibniz Association
                Funded by: German Academic Exchange Service (DAAD)
                Funded by: state of Saxony Anhalt
                Funded by: DFG Graduate Training Center
                Award ID: GRK1026
                Funded by: Leibniz Institute of Plant Biochemistry (IPB)
                Funded by: European Cooperation in Science and Technology (COST)
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                January 2018
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