Abstract
Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (β-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.
Highlights
Posttranslational protein arginylation is the process of ribosome-independent incorporation of arginine from charged tRNA to N-terminal amino acids of proteins or sidechains of internal glutamate or aspartate residues and is mediated by arginyl-tRNA protein transferases (ATE)1
We demonstrated that ATE abundance was increased in moss (Physcomitrella patens) leafy gametophores after application of the stress hormone abscisic acid (ABA), in darkness or in red light, using a translational ATE:GUS fusion at the endogenous locus [11]
We employed this transgenic ATE:GUS line to pull down ATE together with potential interaction partners as well as arginylation targets from tissues where ATE abundance was monitored via histochemical GUS staining
Summary
Minal amidohydrolases (NTAQ, NTAN) [4], generating glutamic acid and aspartic acid, respectively. An increase of arginylation efficiency was observed upon addition of cell lysates in the same study, suggesting the presence of interaction partners or supporting co-factors. To date, no such interaction partners have been identified in any eukaryote, except LIAT1 (ligand of ATE1) from mouse [13]. Whereas the identification of the exact arginylation targets of ATE is pending in plants, the identification of interaction partners of ATE is necessary to fully understand the function of the N-end rule pathway in eukaryotes. We present the first identification of N-terminally arginylated proteins from a model plant by mass spectrometry as well as a novel interaction partner of ATE, an Hsp class I chaperone. On the basis of our findings we distinguish between arginylation targets that interact with ATE to become arginylated and functional interaction partners of ATE that interact with ATE without being targets for arginylation
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