PT  - JOURNAL ARTICLE
AU  - Augustin C. Mot
AU  - Erik Prell
AU  - Maria Klecker
AU  - Christin Naumann
AU  - Frederik Faden
AU  - Bernhard Westermann
AU  - Nico Dissmeyer
TI  - Real-time detection of PRT1-mediated ubiquitination via fluorescently labeled substrate probes
AID  - 10.1101/062067
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 062067
4099  - http://biorxiv.org/content/early/2016/09/04/062067.short
4100  - http://biorxiv.org/content/early/2016/09/04/062067.full
AB  - The N-end rule pathway has emerged as a major system for controlling protein stability in medical, animal and plant sciences as well as agriculture. This included the discovery of novel functions and enzymes of the pathway. Ubiquitination mechanism and substrate specificity of bona fide N-end rule pathway E3 Ubiquitin ligases is still elusive. Taking the first discovered bone fide plant N-end rule E3 ligase PROTEOLY-SIS1 (PRT1) as a model, we describe a novel tool to molecularly characterize polyubiquitinylation live, in real-time. We demonstrate that PRT1 is indeed an E3 ligase and gain mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination can then be measured by rapid in-gel fluorescence scanning in classical end-point assays as well as in real time by fluorescence polarization in standard microplate readers. Enzymatic activity, substrate specificity, reaction mechanisms and optimization can be easily investigated ad hoc in short time and with significantly reduced reagent consumption.