title: Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes.
abstract: 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.
authors.affiliation: Independent Junior Research Group on Protein Recognition and Degradation, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.|ScienceCampus Halle - Plant-based Bioeconomy, Betty-Heimann-Str. 3, Halle (Saale), D-06120, Germany.
authors.forename: Augustin C
authors.initials: AC
authors.lastname: Mot
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Prell
authors.affiliation: Independent Junior Research Group on Protein Recognition and Degradation, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.|ScienceCampus Halle - Plant-based Bioeconomy, Betty-Heimann-Str. 3, Halle (Saale), D-06120, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Klecker
authors.affiliation: Independent Junior Research Group on Protein Recognition and Degradation, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.|ScienceCampus Halle - Plant-based Bioeconomy, Betty-Heimann-Str. 3, Halle (Saale), D-06120, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Naumann
authors.affiliation: Independent Junior Research Group on Protein Recognition and Degradation, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.|ScienceCampus Halle - Plant-based Bioeconomy, Betty-Heimann-Str. 3, Halle (Saale), D-06120, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Faden
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Westermann
authors.affiliation: Independent Junior Research Group on Protein Recognition and Degradation, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, Halle (Saale), D-06120, Germany.|ScienceCampus Halle - Plant-based Bioeconomy, Betty-Heimann-Str. 3, Halle (Saale), D-06120, Germany.
authors.forename: Nico
authors.identifier: 0000-0002-4156-3761
authors.initials: N
authors.lastname: Dissmeyer
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D003199:Computer Systems; D005456:Fluorescent Dyes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism; D059748:Proteolysis; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D029681:Arabidopsis Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C113475:PRT1 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases
keywords: E3 ligases; N-end rule pathway; activity profiling; fluorescent dyes; labeling chemistry; protein labeling; proteolysis; ubiquitination