arginyl-tRNA Protein Transferase Research Articles

Copper catalyzed cycloaddition for the synthesis of non isomerisable 2′ and 3′-regioisomers of arg-tRNAarg

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2′- or 3′-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2′- or 3′-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5′-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5′-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2′ and 3′) which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

Arginyl-tRNA protein transferase 1 (ATE1) regulates protein degradation by Arg/N-end rule pathway

Previous studies have shown that deletion or downregulation of ATE1 can lead to neurodegeneration, behavioral abnormalities, hyperphagia and lower body weight. To determine how loss of ATE1 affects energy metabolism and food intake, ATE1-deficient and ATE1-containing mice were given high fat diet for 12 weeks. Based on our current data, we found significant sex differences in body weight and food intakes, with ATE1-deficient mice less sensitive to the obesogenic effects of high fat diet even while consuming the same amount of high fat diet as controls. Interestingly, ATE1-deficient mice exposed to high fat diet displayed lower plasma leptin levels. Leptin-treated ATE1-deficient mice show changes in pSTAT3 expression in the arcuate nucleus of the hypothalamus relative to leptin-treated ATE1-containing mice. Lastly, diet-induced obesity can be ameliorated by tamoxifen-induced knockout of ATE1 in adult male mice relative to vehicle-treated WT mice. Taken together, these data indicate that loss of ATE1 produces attenuated leptin levels in mouse models of diet-induced obesity and can mitigate the effects of high fat diet. NIGMS, 1SC1GM144190-01 (ESN), NINDS R15NS095317 (CSB), Texas Woman's University internal funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The structural basis of tRNA recognition by arginyl-tRNA-protein transferase

Arginyl-tRNA-protein transferase 1 (ATE1) is a master regulator of protein homeostasis, stress response, cytoskeleton maintenance, and cell migration. The diverse functions of ATE1 arise from its unique enzymatic activity to covalently attach an arginine onto its protein substrates in a tRNA-dependent manner. However, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. Here, we describe the three-dimensional structures of Saccharomyces cerevisiae ATE1 with and without its tRNA cofactor. Importantly, the putative substrate binding domain of ATE1 adopts a previously uncharacterized fold that contains an atypical zinc-binding site critical for ATE1 stability and function. The unique recognition of tRNAArg by ATE1 is coordinated through interactions with the major groove of the acceptor arm of tRNA. Binding of tRNA induces conformational changes in ATE1 that helps explain the mechanism of substrate arginylation.

Arginyl-tRNA-protein transferase 1 (ATE1) promotes melanoma cell growth and migration.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

Arginyl-tRNA-protein transferase 1 contributes to governing optimal stability of the human immunodeficiency virus type 1 core

BackgroundThe genome of human immunodeficiency virus type 1 (HIV-1) is encapsulated in a core consisting of viral capsid proteins (CA). After viral entry, the HIV-1 core dissociates and releases the viral genome into the target cell, this process is called uncoating. Uncoating of HIV-1 core is one of the critical events in viral replication and several studies show that host proteins positively or negatively regulate this process by interacting directly with the HIV-1 CA.ResultsHere, we show that arginyl-tRNA-protein transferase 1 (ATE1) plays an important role in the uncoating process by governing the optimal core stability. Yeast two-hybrid screening of a human cDNA library identified ATE1 as an HIV-1-CA-interacting protein and direct interaction of ATE1 with Pr55gag and p160gag − pol via HIV-1 CA was observed by cell-based pull-down assay. ATE1 knockdown in HIV-1 producer cells resulted in the production of less infectious viruses, which have normal amounts of the early products of the reverse transcription reaction but reduced amounts of the late products of the reverse transcription. Interestingly, ATE1 overexpression in HIV-1 producer cells also resulted in the production of poor infectious viruses. Cell-based fate-of-capsid assay, a commonly used method for evaluating uncoating by measuring core stability, showed that the amounts of pelletable cores in cells infected with the virus produced from ATE1-knockdown cells increased compared with those detected in the cells infected with the control virus. In contrast, the amounts of pelletable cores in cells infected with the virus produced from ATE1-overexpressing cells decreased compared with those detected in the cells infected with the control virus.ConclusionsThese results indicate that ATE1 expression levels in HIV-1 producer cells contribute to the adequate formation of a stable HIV-1 core. These findings provide insights into a novel mechanism of HIV-1 uncoating and revealed ATE1 as a new host factor regulating HIV-1 replication.Graphic abstract

The Ligand of Ate1 is intrinsically disordered and participates in nucleolar phase separation regulated by Jumonji Domain Containing 6

The Ligand of Ate1 (Liat1) is a protein of unknown function that was originally discovered through its interaction with arginyl-tRNA protein transferase 1 (Ate1), a component of the Arg/N-degron pathway of protein degradation. Here, we characterized the functional domains of mouse Liat1 and found that its N-terminal half comprises an intrinsically disordered region (IDR) that facilitates its liquid-liquid phase separation (LLPS) in the nucleolus. Using bimolecular fluorescence complementation and immunocytochemistry, we found that Liat1 is targeted to the nucleolus by a low-complexity poly-K region within its IDR. We also found that the lysyl-hydroxylase activity of Jumonji Domain Containing 6 (Jmjd6) modifies Liat1, in a manner that requires the Liat1 poly-K region, and inhibits its nucleolar targeting and potential functions. In sum, this study reveals that Liat1 participates in nucleolar LLPS regulated by Jmjd6.

Molecular basis for cysteine oxidation by plant cysteine oxidases from Arabidopsis thaliana

Plant Cysteine Oxidases (PCOs) play important roles in controlling the stability of Group VII ethylene response factors (ERF-VIIs) via Arg/N-degron pathway through catalyzing the oxidation of their N-Cys for subsequent Arginyl-tRNA--protein transferase 1 (ATE1) mediated arginine installation. Here we presented the crystal structures of PCO2, PCO4, and PCO5 from Arabidopsis thaliana (AtPCOs) and examined their in vitro activity by Mass spectrometry (MS). On the basis of Tris-bound AtPCO2, we modelled the structure of Cys-bound AtPCO2 and identified key AtPCO2 residues involved in N-Cys recognition and oxidation. Alanine substitution of potential N-Cys interaction residues impaired the activity of AtPCO5 remarkably. The structural research, complemented by mutagenesis and MS experiments, not only uncovers the substrate recognition and catalytic mode by AtPCOs, but also sheds light on the future design of potent inhibitors for plant cysteine oxidases.

Insights on a putative aminoacyl-tRNA-protein transferase of Leishmania major.

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.

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

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.

Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P.patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P.patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

Genome-wide association mapping unravels the genetic control of seed germination and vigor in Brassica napus

Rapid and uniform seed germination is a crucial prerequisite for crop establishment and high yield levels in crop production. A disclosure of genetic factors contributing to adequate seed vigor would help to further increase yield potential and stability. Here we carried out a genome-wide association study in order to define genomic regions influencing seed germination and early seedling growth in oilseed rape (Brassica napus L.). A population of 248 genetically diverse winter-type B. napus accessions was genotyped with the Brassica 60k SNP Illumina genotyping array. Automated high-throughput in vitro phenotyping provided extensive data for multiple traits related to germination and early vigor, such as germination speed, absolute germination rate and radicle elongation. The data obtained indicate that seed germination and radicle growth are strongly environmentally dependent, but could nevertheless be substantially improved by genomic-based breeding. Conditions during seed production and storage were shown to have a profound effect on seed vigor, and a variable manifestation of seed dormancy appears to contribute to differences in germination performance in B. napus. Several promising positional and functional candidate genes could be identified within the genomic regions associated with germination speed, absolute germination rate, radicle growth and thousand seed weight. These include B. napus orthologs of the Arabidopsis thaliana genes SNOWY COTYLEDON 1 (SCO1), ARABIDOPSIS TWO-COMPONENT RESPONSE REGULATOR (ARR4), and ARGINYL-t-RNA PROTEIN TRANSFERASE 1 (ATE1), which have been shown previously to play a role in seed germination and seedling growth in A. thaliana.

Applying Arginylation for Bottom-Up Proteomics.

Arginylation is an enzymatic reaction in which arginyl-tRNA protein transferase 1 (ATE1, EC 2.3.2.8) conjugates a single arginyl moiety from aminoacylated tRNA(Arg) onto a target polypeptide. We established arginylation for in vitro labeling of peptides with N-terminal acidic amino acids. Consistent with prior knowledge, arginylated peptides flanked by basic amino acids result in rich redundant MS/MS fragment spectra using various precursor fragmentation modes. Arginylation carried out by ATE1 is a fast method for labeling peptides. Sequence-specific proteolytic digest of proteins is best carried out using a double digest of proteins by Lys-C and Asp-N to generate peptides with a basic amino acid on the C-terminus and an acidic amino acid on the N-terminus. Under these conditions, arginylation is specific for N-terminal acidic amino acids and results in a near 2× sequence coverage in the MS/MS spectrum are achieved.

Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

RATIONALETandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.METHODSHere we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini.RESULTSWe demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue.CONCLUSIONSWe introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.

Contractility of myofibrils from the heart and diaphragm muscles measured with atomic force cantilevers: Effects of heart-specific deletion of arginyl-tRNA–protein transferase

Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89±9 nN/μm(2)) when compared to WT (132±11 nN/μm(2)), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K(act)), redevelopment (K(tr)), and relaxation (K(rel)). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209±31 nN/μm(2)) when compared to WT (123±20 nN/μm(2)). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K(act), but α-MHCAte1 myofibrils presented a faster K(rel) (6.11±0.41s(-1) vs 4.63±0.41 s(-1)). Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.

Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding

Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca2+ conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40°C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress.We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.

Arginylated Calreticulin at Plasma Membrane Increases Susceptibility of Cells to Apoptosis

Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a preapoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase are less susceptible to apoptosis than wild type cells. Under these conditions R-CRT is present on the cell membrane but at early stages is differently localized in stress granules. Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able to both increase the number of cells undergoing apoptosis in wild type cells and overcome apoptosis resistance in cells lacking arginyl-tRNA protein transferase that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function, and survival.

Heme regulates protein homeostasis at transcription, protein translation, and degradation levels

Heme, as a prosthetic group of proteins, is an iron-protoporphyrin involved in a wide range of cellular functions. Cellular heme levels vary due to the accurate balance of its synthesis and degradation. The “heme sensor protein” is currently a focus of investigation because heme has been found as a cellular signaling messenger involved in various biologic processes, including gene expression, protein localization, protein stability and microRNA processing. Several eukaryotic transcriptional factors can be regulated by heme, including heme activator protein (Hap1), Bach1, REV-erbα, and neuronal PAS domain protein 2 (NPAS2). Especially, the two circadian transcriptional factors serving as the heme sensor, REV-erbα and NPAS2, coordinate the circadian clock with metabolic pathways. It is well established that heme regulates the activity of heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), which serves as a feedback inhibitor of protein translation in both erythroid and non-erythroid cells. Additionally, heme is involved in protein degradation by inducing the degradation of several proteins such as the iron response regulator (Irr), iron regulatory protein 2 (IRP2), Bach1, and circadian factor period 2 (Per2). The N-end rule ubiquitin-dependent protein degradation pathway has also been identified as a sensor of heme, which blocks the function of arginyl-tRNA protein transferase (ATE1) and E3 ubiquitin ligase. In this review, we summarize the regulatory roles of heme at the levels of transcription, protein translation, and protein degradation, highlighting the role of heme in maintaining cellular homeostasis.

The arginylation-dependent association of calreticulin with stress granules is regulated by calcium

Post-translational modifications of proteins are important for the regulation of cell functions; one of these modifications is post-translational arginylation. In the present study, we show that cytoplasmic CRT (calreticulin) is arginylated by ATE1 (arginyl-tRNA protein transferase). We also show that a pool of CRT undergoes retrotranslocation from the ER (endoplasmic reticulum) to the cytosol, because in CRT-knockout cells transfected with full-length CRT (that has the signal peptide), cytoplasmic CRT appears as a consequence of its expression and processing in the ER. After the cleavage of the signal peptide, an N-terminal arginylatable residue is revealed prior to retrotranslocation to the cytoplasm where arginylation takes place. SGs (stress granules) from ATE1-knockout cells do not contain CRT, indicating that CRT arginylation is required for its association to SGs. Furthermore, R-CRT (arginylated CRT) in the cytoplasm associates with SGs in cells treated with several stressors that lead to a reduction of intracellular Ca2+ levels. However, in the presence of stressors that do not affect Ca2+ levels, R-CRT is not recruited to these loci despite the fact that SGs are formed, demonstrating Ca2+-dependent R-CRT association to SGs. We conclude that post-translational arginylation of retrotranslocated CRT, together with the decrease in intracellular Ca2+, promotes the association of CRT to SGs.

The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development

The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. This proteolytic system is present in all organisms examined and has been shown to have a multitude of functions in animals and fungi. In plants, however, the functional understanding of the N-end rule pathway is only beginning. The N-end rule has a hierarchic structure. Destabilizing activity of N-terminal Asp, Glu, and (oxidized) Cys requires their conjugation to Arg by an arginyl-tRNA-protein transferase (R-transferase). The resulting N-terminal Arg is recognized by the pathway's E3 ubiquitin ligases, called "N-recognins." Here, we show that the Arabidopsis R-transferases AtATE1 and AtATE2 regulate various aspects of leaf and shoot development. We also show that the previously identified N-recognin PROTEOLYSIS6 (PRT6) mediates these R-transferase-dependent activities. We further demonstrate that the arginylation branch of the N-end rule pathway plays a role in repressing the meristem-promoting BREVIPEDICELLUS (BP) gene in developing leaves. BP expression is known to be excluded from Arabidopsis leaves by the activities of the ASYMMETRIC LEAVES1 (AS1) transcription factor complex and the phytohormone auxin. Our results suggest that AtATE1 and AtATE2 act redundantly with AS1, but independently of auxin, in the control of leaf development.

Post-translational Arginylation of Calreticulin: A NEW ISOSPECIES OF CALRETICULIN COMPONENT OF STRESS GRANULES

Post-translational arginylation consists of the covalent union of an arginine residue to a Glu, Asp, or Cys amino acid at the N-terminal position of proteins. This reaction is catalyzed by the enzyme arginyl-tRNA protein transferase. Using mass spectrometry, we have recently demonstrated in vitro the post-translational incorporation of arginine into the calcium-binding protein calreticulin (CRT). To further study arginylated CRT we raised an antibody against the peptide (RDPAIYFK) that contains an arginine followed by the first 7 N-terminal amino acids of mature rat CRT. This antibody specifically recognizes CRT obtained from rat soluble fraction that was arginylated in vitro and also recognizes endogenous arginylated CRT from NIH 3T3 cells in culture, indicating that CRT arginylation takes place in living cells. Using this antibody we found that arginylation of CRT is Ca2+-regulated. In vitro and in NIH 3T3 cells in culture, the level of arginylated CRT increased with the addition of a Ca2+ chelator to the medium, whereas a decreased arginine incorporation into CRT was found in the presence of Ca2+. The arginylated CRT was observed in the cytosol, in contrast to the non-arginylated CRT that is in the endoplasmic reticulum. Under stress conditions, arginylated CRT was found associated to stress granules. These results suggest that CRT arginylation occurs in the cytosolic pool of mature CRT (defined by an Asp acid N-terminal) that is probably retrotranslocated from the endoplasmic reticulum.