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

Abstract

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.

N‐terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N‐end rule pathway

“…Arabidopsis has two characterised E3 ligases that recognise different types of destabilising residues. PROTEOLYSIS1 (PRT1) recognises aromatic Nt amino acids, whereas PRT6 is specific for basic Nt residues (Potuschak et al, 1998;Stary et al, 2003;Garz on et al, 2007;Graciet et al, 2010;Mot et al, 2018). As well as primary destabilising residues revealed by endopeptidase cleavage, PRT6 substrates can be generated via enzymatic modification of secondary and tertiary destabilising residues (Figs 1, S1).…”

The plant N‐degron pathways of ubiquitin‐mediated proteolysis

“…However, a screen in Arabidopsis to identify genetic components responsible for degradation of substrates with Nt‐Phe identified PROTEOLYSIS (PRT)1, a protein with a unique combination of structural domains, comprising two RING finger and one ZZ domain (Bachmair et al ; Potuschak et al ) (Figure B). Further analysis showed that PRT1 acts as a ubiquitin ligase and only recognizes substrates with Nt‐aromatic (Phe‐, Tyr‐ and Trp‐) residues (Stary et al ; Mot et al ). This indicated that the ClpS‐like domain function of UBR1 has been replaced in plants with a novel domain‐combination acting within a separate ligase.…”

Life and death of proteins after protease cleavage: protein degradation by the N‐end rule pathway

“…Moreover, an N-terminal destabilizing residue is not necessarily sufficient for the generation of an N-degron. Indeed, the accessibility of the N-terminal residue for N-recognin binding, the properties of the residues neighboring the N-terminus and the proximity of a Lys residue that may be ubiquitylated are also important (Tasaki et al, 2012;Wadas et al, 2016;Mot et al, 2018). The complexity of an N-degron probably explains why many proteins with a presumed N-terminal destabilizing residue appear to be relatively abundant and stable in plant cells (Li et al, 2017).…”

“…In vivo studies with artificial N‐end rule reporter substrates in plants show that PRT 6 in specific for positively charged or Type 1 residues (Garzon et al ., ) and that PRT 1 recognizes aromatic hydrophobic or Type 2 residues (Potuschak et al ., ). Furthermore, in vitro ubiquitylation assays of fluorescently labeled artificial N‐end rule substrates confirmed the specificity of PRT 1 and its E3 ligase activity (Mot et al ., ). Note that the degradation of proteins with N‐terminal Met, as well as through the Ac/ and Pro/N‐end rule (all represented on a beige background) have not yet been demonstrated to exist in plants, but are found in yeast (Hammerle et al ., ; Chen et al ., ).…”