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

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          Summary

          • 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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          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
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            New insights into ubiquitin E3 ligase mechanism.

            E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated.
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              Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants

              Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by root waterlogging or foliage submergence, triggers changes in gene transcription and mRNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production 1 . In contrast to animals 2 , oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen in Arabidopsis, through its regulation of key hypoxia response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated Ethylene Response Factor (ERF) Group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the N-terminus initiating with MetCys- (MC-). Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice 3 , was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, suggesting that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses 4 .
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                Author and article information

                Contributors
                nico.dissmeyer@ipb-halle.de
                Journal
                New Phytol
                New Phytol
                10.1111/(ISSN)1469-8137
                NPH
                The New Phytologist
                John Wiley and Sons Inc. (Hoboken )
                0028-646X
                1469-8137
                09 March 2017
                January 2018
                : 217
                : 2 ( doiID: 10.1111/nph.2018.217.issue-2 )
                : 613-624
                Affiliations
                [ 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@ 123456ipb-halle.de

                Author information
                http://orcid.org/0000-0002-4156-3761
                Article
                NPH14497
                10.1111/nph.14497
                5763331
                28277608
                a376ea97-15a1-4296-a7ab-a7665f3530d0
                © 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.

                History
                : 05 December 2016
                : 26 January 2017
                Page count
                Figures: 3, Tables: 0, Pages: 12, Words: 9987
                Funding
                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)
                Categories
                Full Paper
                Research
                Full Papers
                Custom metadata
                2.0
                nph14497
                January 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.2.8 mode:remove_FC converted:11.01.2018

                Plant science & Botany
                activity profiling,e3 ligases,fluorescent dyes,labeling chemistry,n‐end rule pathway,protein labeling,proteolysis,ubiquitination

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