Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Majovsky, Petra; Naumann, Christin; Lee, Chil-Woo; Lassowskat, Ines; Trujillo, Marco; Dißmeyer, Nico; Hoehenwarter, Wolfgang

doi:10.1021/pr500164j

Cited by 47 publications

(51 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is astonishing, although we cannot fully exclude that the antibodies used in this study resulted in a biased sampling of arginylated proteins due to their specificity to N-terminal RE or RD instead of being only specific for N-terminal Arg, similar to those used in (23). Additionally, it remains unknown why other proteomic approaches (31,32) failed to detect arginylation events in plants that did not correspond to the hierarchical order of the N-end rule pathway. Thus, we suggest that other functions of arginylation besides targeting for degradation may not be present in moss or at least not to the extent observed in mammals.…”

Section: Resultsmentioning

confidence: 87%

“…5B). Although both hypotheses need experimental verification, the interaction of ATE with a small HSP emphasizes the fact that an integral feature of protein stability within the N-end rule pathway may not solely be the N-terminal amino acid of a protein, but also its accessibility (31). This is already evident in the second branch of the N-end rule pathway, the Ac/N-end rule pathway, which is present in yeast and mammals but is also proposed to exist in plants (8 -10, 78).…”

Section: Resultsmentioning

confidence: 99%

“…Unacetylated N-terminal methionine can further act as primary destabilizing residue in certain cases (99) but is not depicted in the present figure. sensors via their N-terminal cysteine residues (17,18, reviewed in (2)). A few more potential arginylation targets were proposed based on quantitative mass spectrometry including enrichment of N-termini (31,32). However, corresponding arginylated peptides were not identified in these studies.…”

mentioning

confidence: 98%

See 2 more Smart Citations

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Hoernstein

Mueller

Fiedler

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

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 arginyltRNA 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 (␤-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Fö rster 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.

show abstract

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

See 1 more Smart Citation

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Hoernstein

Mueller

Fiedler

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Thus far, studies by other laboratories have failed to find evidence for either a significant arginylation of non-canonical N-terminal amino acid residues or a significant arginylation of internal residues (85,(97)(98)(99).…”

Section: B7abmentioning

confidence: 99%

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Wadas

Piatkov

Brower

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

N␣ -terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified ("canonical") residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only ␣-amino groups of N-terminal residues but also ␥-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.The N-end rule pathway is a set of intracellular proteolytic systems whose unifying feature is the ability to recognize and polyubiquitylate proteins containing N-terminal (Nt) 2 degradation signals called N-degrons, thereby causing the processive degradation of these proteins by the proteasome (Fig. 1, A and B) (1-13). Recognition components of the N-end rule pathway are called N-recognins. In eukaryotes, N-recognins are E3 ubiquitin (Ub) ligases that can target N-degrons. Some N-recognins contain several substrate-binding sites and thereby can recognize (bind to) not only N-degrons but also specific internal (non-N-terminal) degradation signals (Fig. 1A) (14 -18). The main determinant of a protein's N-degron is either an unmodified or chemically modified N-terminal residue. Another determinant of an N-degron is an internal Lys residue(s). It functions as a site of protein polyubiquitylation, is often engaged stochastically (in competition with other "eligible" lysines), and tends to be located in a conformationally disordered region (2,9,19,20). Bacteria also contain the N-end rule pathway, but Ub-independent versions of it (21-26).Regulated degradation of proteins and their natural fragments by the N-end rule pathway has been s...

show abstract

“…This type of MS experiment relies on acquisition of several (usually 5-10, or up to 20 in modern instruments) MS/MS spectra of the most intense signals observed in a so-called survey MS scan (which typically takes from 20 to 500 ms, depending on the instrument) (Majovsky et al, 2014). Thus, the number of MS/MS scans acquirable during the time of the whole DDA experiment is limited and usually essentially lower than the number of peptides detected on the MS level.…”

Section: Gel-free (Lc-based) Bottom-up Proteomic Strategymentioning

confidence: 99%

Mining seed proteome: from protein dynamics to modification profiles

Frolov¹,

Мамонтова²,

Ihling³

et al. 2018

BioComm

Self Cite

View full text Add to dashboard Cite

Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Cited by 47 publications

References 46 publications

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Mining seed proteome: from protein dynamics to modification profiles

Contact Info

Product

Resources

About