Abstract
According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719).
Highlights
Public microarray data indicated that PRT6, ATE1 and ATE2 transcripts were expressed at a low level throughout the plant (Supporting Information Fig. 3)
These results indicate that PRT6 is active in roots, cotyledons and leaves but that removal of PRT6 or ATE1/2 was insufficient to stabilise MC-GUS in cotyledons
We explored the role of the Arg/N-end rule on the Arabidopsis root proteome
Summary
In eukaryotic cells, controlled degradation plays a pivotal role in determining protein abundance throughout signalling and development [1]. Proteins are covalently modified by addition of chains of ubiquitin molecules to internal lysine residues, tagging them for degradation by the 26S proteasome. This reaction is catalysed by E3 ligases, which determine selectivity by recognising a degradation signal (known as the degron) in a protein substrate. The amino-terminal (Nt) residue of a protein was one of the first classes of degrons to be identified, leading to the discovery of the “N-end rule,” which states that the half-life of a protein is determined by its Nt amino acid [2].
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