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. 2015 Jul;15(14):2447-57.
doi: 10.1002/pmic.201400530. Epub 2015 Apr 17.

Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots

Hongtao Zhang  1   2 , Michael J Deery  2 , Lucy Gannon  1 , Stephen J Powers  3 , Kathryn S Lilley  2 , Frederica L Theodoulou  1
Affiliations

Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots

Hongtao Zhang et al. Proteomics. 2015 Jul.

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).

Keywords: N-end rule; Plant proteomics; Quantitative proteomics; Root; TAILS; TMT.

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Figures

Figure 1
Figure 1
The Arg/N-end rule is active in Arabidopsis roots. (A) Schematic representation of the PRT6 branch of the Arg/N-end rule in Arabidopsis. Proteins enter the pathway following cleavage by endopeptidases (EPases) or methionine aminopeptidases (MetAPs) in the case of Met-Cys proteins. C* indicates oxidized Cys, catalyzed by plant cysteine oxidases (PCO). (B) Expression of R-GUS protein stability reporter in Col-0 and prt6 seedlings. In planta, the fusion protein is cleaved by ubiquitin-specific proteases (indicated by arrow) to remove dihydrofolate reductase-ubiquitin (DHFR-Ub), generating a variant of GUS that is preceded by an unstructured region (light gray) with an Nt R residue. From left to right: whole seedling (scale bar: 0.5 cm); leaf/cotyledon axil, root, cotyledon (scale bars: 200 μm). (C) Expression of MC-GUS in 5 day old seedlings of prt6 and Col-0 and MA-GUS in Col-0. Met1 is removed cotranslationally by MetAPs, as indicated by the arrow. The MC-GUS/prt6 line was back-crossed to Col-0 to enable a direct comparison of the same transgene event in wild-type and mutant backgrounds. From left to right: whole seedling (scale bar: 0.5 cm); root (scale bar: 50 μm); cotyledon (scale bar: 200 μm). Cartoons show schematics of DNA constructs.
Figure 2
Figure 2
The Arg/N-end rule does not cause major perturbations in the proteome of roots. (A) Schematic representation of the TMT workflow. A label-swap was performed for the second experiment. (B) Venn diagram showing proteins identified and quantified in two independent experiments. (C) Plots of changes in protein abundance in N-end rule mutants, prt6 and ate1/2 relative to Col-0. Plots depict log (2) transformed ratios of abundance of 3765 proteins ranked from low to high. (D) Scatter plot of the log (2) transformed ratios (prt6: Col-0 versus ate1/2: Col-0) for 3765 proteins. See Table 1 and Supporting Information Table 1 for list of proteins.
Figure 3
Figure 3
Isolation of N-terminal peptides with dimethyl-TAILS. (A) Schematic representation of the TAILS workflow. Primary amines of proteins with free N-termini (star) and lysine (K) side-chain amines of proteins were chemically modified by isotopically distinct dimethyl labelling (light/intermediate/heavy). After combining labelled samples from WT and N-end rule mutant plants, proteins were digested and internal peptides removed via HPG-ALD polymer binding of the free N-terminal amine group. The unbound peptides (highly enriched for N-terminal peptides) were then analysed and quantified by high-accuracy LC-MS/MS. Mascot and ProteomeDiscoverer™ were used for protein identification and quantification. Grey pentagons represent naturally blocked (acetylated) N-termini. (B) Numbers of unique N-terminal (Nt) acetylated (Ac) and non-Nt peptides identified before (pre-TAILS) and after enrichment by TAILS. (C) Venn diagram showing overlap of Ac Nt peptides identified in pre-TAILS and TAILS samples.
Figure 4
Figure 4
Analysis of unique N-terminal peptides. The dataset is restricted to unique peptides with Nt acetylation or dimethylation and available position information. (A) Pie chart showing the total free and acetylated (Ac) unique N-terminal peptides identified. (B–D) Analysis of first and second residues of neo-N-termini and acetylated N-termini. Nt peptides that initiate at amino acid residue > = 3, relative to the translated protein are designated as “other.” (E) Occurrence of different N-terminal amino acid residues in neo-N-terminal peptides. Only neo-N-terminal peptides where the N-terminus corresponds to residue ≥ 3 of the predicted translated protein were analysed. (F) Percentage of proteins with neo-N-termini in different subcellular locations. Subcellular localisation was assigned based on established annotation or TargetP prediction where annotation was lacking. Mito, mitochondrion; perox, peroxisome; dual, dual-targeted to plastid and mitochondrion; sec/ER, secretory pathway/endoplasmic reticulum.
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