Real-time detection of PRT1-mediated ubiquitination via fluorescently labeled substrate probes

SUMMARY The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turn-over in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway were discovered, ubiquitination mechanism and substrate specificity of N-end rule pathway E3 Ubiquitin ligases remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we use a novel tool to molecularly characterize polyubiquitination live, in real-time. We gained mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by 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. Enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc in short time and with significantly reduced reagent consumption. We demonstrated for the first time that PRT1 is indeed an E3 ligase, which was 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.