Molecular characterization of a gene encodingN-myristoyl transferase (NMT) from Triticumaestivum (bread wheat)

Tim Dumonceaux, Raju V.S. Rajala, Rajendra Sharma, Gopalan Selvaraj,and Raju Datla

Abstract: Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in somecases myristoylation is essential to cell growth and survival. We report the cloning of a full-length cDNA encodingNMT from Triticum aestivum (TaNMT). The cDNA included a predicted open reading frame of 1317 nucleotides,which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activ-ity. The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassinga wide array of taxonomic groups. Among the experimentally validated NMTs, TaNMT was most similar to that ofArabidopsis thaliana. Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copygene, with one copy per haploid genome. We expressed TaNMT in Escherichia coli cells and determined that the re-combinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putativelymyristoylated proteins from plants and animals without a strong preference for the plant peptides. TaNMT is the sec-ond experimentally validated plant NMT sequence and the first from a monocotyledonous species.

Key words: N-myristoyl transferase, myristoylation, protein modification, wheat, plant development.

Résumé : La myristoyl-CoA : protéine N-myristoyltransférase (NMT; EC 2.3.1.97) est responsable de l'acylation de laglycine la plus proche de la méthionine N-terminale par l'ajout d'un acide myristique après enlèvement de la méthio-nine chez certaines protéines eucaryotes. Dans certains cas, cette myristoylation est essentielle à la croissance et à lasurvie de la cellule. Les auteurs rapportent le clonage d'un ADNc complet codant pour une NMT chez le Triticum aes-tivum (TaNMT). Cet ADNc comprend une région codant prédite de 1317 nucléotides, laquelle coderait pour une pro-téine de 438 acides aminés dont tous ceux connus comme étant importants pour l'activité NMT. Les séquencenucléotidiques et peptidiques de TaNMT ont été comparées à celles codant pour des NMT chez 14 autres espèces ap-partenant à une vaste gamme de groupes taxinomiques. Parmi les NMT qui ont fait l'objet d'une validation expérimen-tale, c'est celle de l'Arabidopsis thaliana qui était la plus semblable à TaNMT. Une analyse Southern de l'ADNgénomique du blé a révélé que TaNMT est codée par un gène simple copie présent en une seule copie par génome ha-ploïde. Les auteurs ont exprimé TaNMT chez Escherichia coli et montré que la protéine recombinante possède une ac-tivité NMT et catalyse la N-myristoylation de peptides provenant de protéines animales ou végétales connues commeétant myristoylées, bien qu'ayant une forte préférence pour les peptides végétaux. TaNMT est la seconde séquence deNMT d'origine végétale et la première chez une monocotylédone.

Mots clés : N-myristoyltransférase, myristoylation, modification des protéines, blé, développement des plantes.

[Traduit par la Rédaction] Dumonceaux et al. 1042

Introduction

In eukaryotes, a subset of cellular proteins are either co-or post-translationally modified by addition to the N-

terminus of a myristic acid (C14:0) moiety. This is catalyzedby myristoyl-CoA:protein N-myristoyl transferase (NMT).NMT acts on a wide range of proteins, which generally pos-sess an N-terminal myristoylation motif, (M)GNXXXXRR

Genome 47: 1036–1042 (2004) doi: 10.1139/G04-074 © 2004 NRC Canada

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Received 27 April 2004. Accepted 9 June 2004. Published on the NRC Research Press Web site at http://genome.nrc.ca on26 November 2004.

Corresponding Editor: G.J. Scoles.

T. Dumonceaux, G. Selvaraj, and R. Datla.1 National Research Council of Canada, Plant Biotechnology Institute, 110Gymnasium Place, Saskatoon, SK S7N 0W0, Canada.R.V.S. Rajala. Department of Cell Biology and Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City,OK 73104, USA.R. Sharma. Saskatoon Cancer Centre, University of Saskatchewan, Saskatoon, SK S7N 4H4, Canada.

1Corresponding author (e-mail: raju.datla@nrc-cnrc.gc.ca).

(Thompson and Okuyama 2000) but have few other com-mon structural features. For myristoylation to occur the N-terminal methionine residue must be removed by methionineaminopeptidase (Arfin et al. 1995). In animal and fungalcells, protein myristoylation has been observed in proteinsinvolved in signal transduction and cellular signalling, in-cluding several oncoproteins, the Gα subunit, and proteinsinvolved in ADP ribosylation (Rajala et al. 2000). N-myristoylation is crucial to cell survival, as mutations inNMT cause recessive lethality in yeast and in Drosophila(Duronio et al. 1989; Ntwasa et al. 2001). NMT-encodingcDNAs have been isolated and (or) NMT proteins purifiedand characterized from several animal and fungal cells, in-cluding human (Giang and Cravatt 1998), mouse (Giang andCravatt 1998), and bovine tissues (Raju et al. 1994; Rajalaand Sharma 1995; Raju et al. 1997), Saccharomycescerevisiae (Zhang et al. 1996), and Arabidopsis thaliana (Qiet al. 2000). The biochemical characteristics of the nativeand recombinant enzymes have been reviewed (Boutin 1997;Rajala et al. 2000; Farazi et al. 2001).

In plants, the theme appears to be similar, in that manyknown or putatively myristoylated proteins are involved incellular signalling. These include CDPKs (calcium-dependent, calmodulin-independent protein kinases) in awide range of plants, Fen/Pto protein kinases, phospholipaseDγ, the Gα subunit, and the myristoylated alanine-rich Ckinase substrate (MARCKS) protein (Thompson andOkuyama 2000; Lu and Hrabak 2002; Dammann et al.2003). The phytopathogen Pseudomonas syringae uses thehost NMT to target its avirulence (Avr) proteins to the hostmembrane, where they contribute to pathogen virulence indisease-susceptible plants (Nimchuk et al. 2000). In spite ofthe clear importance of NMT activity to plant growth, devel-opment, and disease susceptibility, only 1 plant NMT hasbeen cloned and extensively characterized, that ofArabidopsis thaliana (AtNMT) (Qi et al. 2000). This NMT,although very similar to the mammalian enzymes in its roleand its selectivity for myristoyl-CoA as a substrate, is only~50% identical at the primary structure level to the mamma-lian and fungal NMTs. In addition, the plant enzyme moreefficiently catalyzes the N-terminal myristoylation of pep-tides derived from plant than from mammalian proteins.Like mammalian NMTs, AtNMT is expressed ubiquitouslyand at higher levels in developmentally active tissues, and isessential for proper growth and development (Qi et al.2000). Although proteins with myristoylation motifs arewidely distributed throughout the plant kingdom, there havebeen no other plant NMTs identified except for partially pu-rified NMT activity in Triticum aestivum (bread wheat)(Heuckeroth et al. 1989). Thus, there is no information onthe structural and functional diversity of plant NMTs.

Wheat, which is one of the most important crops grown inthe world today, has been subject to extensive molecular char-acterization and is represented in the publicly available data-bases by more than 550 000 expressed sequence tags (ESTs)(http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html) ,the most of any plant species. Many ESTs for wheat and forother organisms are annotated only by their BLAST (basiclocal alignment search tool) similarities and have not beenassigned validated functions. We here describe the cloningof a full-length cDNA encoding NMT from wheat (Triticum

aestivum ‘Fielder’; TaNMT), the expression of therecombinant enzyme in E. coli, and validation of its enzy-matic activity.

Materials and methods

Isolation of a cDNA encoding TaNMTA single 597-nucleotide EST (GenBank accession No.

BE402458) with similarity to AtNMT by BLASTx was foundin the International Triticeae EST Cooperative database(ITEC; http://wheat.pw.usda.gov/genome, as of 16 March2000, 16 372 sequences). This apparently 5′ truncated se-quence was used to design primers for RT-PCR (5′-TCA-ACTTCCTCTGCGTCC-3′ and 5′-TGTCCACCACATCCTCCT-3′). Total RNA was isolated from wheat (Triticumaestivum ‘Fielder’) leaves and used as a template for Super-script II RT (Invitrogen Canada, Burlington, Ont.) witholigo-dT as primer to generate first-strand cDNA. This wasused for PCR with the primers described (500 nM each) and1 U Taq polymerase (Invitrogen Canada, Burlington, Ont.)under the following conditions: 94 °C, 2 min, followed by30 cycles at 94 °C, 30 s; 55 °C, 30 s; and 72 °C, 1 min. The417-bp product was cloned into pCR2.1 (Invitrogen Canada,Burlington, Ont.) according to the manufacturer’s recom-mendations, and the insert was sequenced to verify its iden-tity. To isolate a full-length cDNA corresponding to TaNMT,a cDNA library was prepared from pooled leaf, root andhypocotyl poly(A)+ RNA (‘Fielder’) using a λZAP cDNAsynthesis system (Stratagene, La Jolla, Calif.). The cDNAwas cloned into the UniZAP XR vector (Stratagene) andpackaged (Giga Pack Gold packaging extracts, Stratagene).The library was screened using standard methodology(Ausubel et al. 1995) with the 417-bp RT-PCR product (32P-labelled) as a probe.

Southern blotGenomic DNA was isolated using the method of

Dellaporta (1994) from the leaves of several varieties ofwheat containing various genome complements: T. aestivum‘Fielder’, AABBDD; T. turgidum ‘Sceptre’, AABB;T. urartu ‘Nigram’, AA; T. tauschii ‘China’, DD. GenomicDNA samples (15 µg) were digested with HindIII, EcoRI, orBamHI at 37 °C for 8 h, then submitted to electrophoresis ona 0.8% agarose gel in Tris–acetate–EDTA (40 mmol/L Tris–HCl, 20 mmol/L acetic acid, 1 mmol/L EDTA). The gel wasblotted to Zeta Probe membrane (Bio-Rad Laboratories,Mississauga, Ont.) for 5 h using 0.4 N NaOH as the transfersolution, and the blot was rinsed and dried. Hybridizationwas done overnight at 42 °C in UltraHyb hybridization solu-tion (Ambion Inc., Austin, Tex.) with 1.25 × 106 countsper min/mL of the 32P-labelled probe described earlier. Themembrane was washed twice in 2× SSC + 0.1% SDSat room temperature (1× SDS is 0.15 mol/L NaCl +0.015 mol/L sodium citrate), then twice in 0.1× SSC + 0.1%SDS at 65 °C. Autoradiographs were exposed for 2 d at –70 °C.

Expression of recombinant TaNMT in Escherichia coli,functional assays, and kinetics

A recombinant construct containing the open readingframe (ORF) of TaNMT was prepared in the vector pET28a

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(Novagen Inc., Madison, Wis.). The longest TaNMT cDNAisolated from the library was used as a template in a PCRwith primers designed to amplify only the ORF with EcoRI(5′) and HindIII (3′) recognition sites added to facilitate clon-ing. The recombinant construct was used to transformE. coli BL21 codon plus cells (Stratagene) according to themanufacturer’s recommendations. The recombinant TaNMTprotein was purified from the cytosolic fraction of an E. colilysate according to the manufacturer’s specifications. NMTassays were performed as described (Qi et al. 2000) using apanel of peptide substrates derived from putativelymyristoylated plant proteins and known myristoylated mam-malian proteins as described in Table 1.

Results and discussion

The deposition of enormous volumes of EST data intopublicly available databases is an ongoing and useful effort.An excellent interface is available to query the more than

550 000 wheat ESTs that are currently publicly available atGrain Genes (http://wheat.pw.usda.gov/index.html). How-ever, the functional assignments for many of these ESTs arelimited to BLAST scores indicating similarity to known orunknown proteins. In some cases, BLAST similarities canbe misleading; for example, in Arabidopsis, of two genesthat are annotated as NMT-like (chromosomes 2 and 5),only NMT1 (chromosome 5) has demonstrable NMT activ-ity (Qi et al. 2000). We have experimentally validated theidentity of a cDNA encoding myristoyl-CoA:protein N-myristoyl transferase from wheat (TaNMT), the first from amonocot species.

To clone the TaNMT cDNA, we produced a cDNA libraryfrom pooled wheat polyA+ RNA. We tried to use the full-length AtNMT cDNA as a probe to screen this library butwere unsuccessful after several attempts at hybridization un-der low-stringency conditions. We then found that the se-quence repository from wheat tissues provided by theInternational Triticeae EST Cooperative included a clone

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Peptide sequence Peptide ID Specific activitya, pmol/min/mg

No peptide 78GSSKSKPKR Src 3295 (1420)GNAAAAKKRR A kinase 5789 (1330)GHRHSKSKKK Arabidopsis CDPK6 5196 (25 000)AHRHSKSKKK Arabidopsis CDPK6 G2A 135MGHRHSKSKK Arabidopsis CDPK6 M 97GGCFSKKYRR Tobacco CDPK1 2470 (980)GLLCSRSRRR Arabidopsis Gα GPA1 7008GQCCSRAPKK Zea mays CDPK9 1194GICLSAQVKK Arabidopsis APK1 1316GCFHSKAAKK Arabidopsis CNB-like protein 3175GCASSLPDRR Arabidopsis ARA6 (Rab GTPase) 1207GSKYSKATRR Tomato Fen kinase 5372 (19 000)GSSCSRSHRR Rice Gα 3302

Note: TaNMT, myristoyl-CoA:protein N-myristoyl transferase of Triticum aestivum.aValues in parentheses indicate the specific activity of recombinant AtNMT on the same substrates (Qi et al. 2000).

Table 1. Specific activity of recombinant TaNMT on various peptide substrates of plant and mamma-lian origin.

Organism Abbreviation Type GenBank ID Reference

Triticum aestivum (wheat) TaNMT Plant AY489191 This workArabidopsis thaliana AtNMT Plant AF193616 Qi et al. 2000Oryza sativa (rice)a OsNMT Plant NM_191679 Sasaki et al. 2002Brassica oleraceaa BoNMT Plant AF180355 Wroblewski et al. 2000Homo sapiens (human) HsNMT Animal AF043324 Giang and Cravatt 1998Mus musculus (mouse) MmNMT Animal AF043326 Giang and Cravatt 1998Drosophila melanogaster DmNMT Animal AF053725 Ntwasa et al. 1997Bos taurus (bovine) BtNMT Animal Raju et al. 1994Histoplasma capsulatum HcNMT Fungal L25118 Lodge et al. 1994Saccharomyces cerevisiae ScNMT Fungal M23726 Duronio et al. 1989Filobasidiella neoformans FnNMT Fungal L25116 Lodge et al. 1994Emericella nidulans EnNMT Fungal AY057437 Shaw et al. 2002Aspergillus fumigatus AfNMT Fungal AB035414 unpublishedCandida albicans CaNMT Fungal M80544 Wiegand et al. 1992Leishmania major LmNMT Trypanosome AF305956 Unpublished

Note: NMT, myristoyl-CoA:protein N-myristoyl transferase.aPutative NMTs; annotation based on BLAST analysis of DNA sequence information.

Table 2. Sources of the NMT sequences used in this study.

with similarity to AtNMT by BLASTx, and used that se-quence as a template for primer design in order to generate ahomologous probe for the cDNA library. A total of 3 × 105

plaque-forming units was screened with this 417-bp probe,and 9 hybridizing clones were purified through additionalrounds of screening. Three clones containing an apparentlyfull-length cDNA were isolated with an ORF of 1317 nt, a 5′UTR of 117 nt, and a 3′ UTR (untranslated region) of 270 nt(Gen Bank acc. no. AY489191 for TaNMT). The longestORF in each clone contained a start codon in the context ofa consensus sequence (GCCATGGCCG) that is favorablefor translation in monocots (Baga et al. 1999).

The ORF encoded a predicted protein of 438 amino acidswith a high degree of sequence similarity to reported mam-malian, plant, and fungal NMTs. TaNMT was comparedwith 14 other known or putative (i.e., annotated according toBLAST similarity) NMTs (Table 2). Among the experimen-tally validated NMTs, TaNMT was most similar in aminoacid sequence to AtNMT (73% identity, 85% similarity us-ing the BLOSUM 62 scoring table); however, among theknown and putative NMTs, TaNMT was most similar toOsNMT (93% identity, 96% similarity), as expected fromthe closer phylogenetic relation of wheat and rice. Analysisof the phylogenetic relations of all the NMT amino acid andnucleotide sequences showed that the clustering pattern re-flected the taxonomic distribution of the source organisms(Fig. 1). Thus, the plant NMTs formed a group, with a splitbetween the monocot-derived (TaNMT, OsNMT) and dicot-derived (AtNMT, BoNMT) sequences. Similarly, animalNMT sequences clustered together, with the mammalian se-quences being distinct from the insect sequence (DmNMT).The three major groups noted among the fungi are consistentwith the three major phylogenetic groups represented: yeast(ScNMT and CaNMT), ascomycetes (EnNMT, AfNMT, andHcNMT), and basidiomycetes (FnNMT). While the basidio-mycete amino acid sequence was most closely related to thetrypanosome (LmNMT) sequence (Fig. 1A), its nucleotidesequence formed a separate group (Fig. 1B).

Multiple sequence alignment of the amino acid sequencesrevealed that TaNMT contains all the residues reported to beimportant for NMT activity, including 2 NMT signature se-quences, (ED)(IV)NFLCXHK (PROSITE accession no.PS00975; aa 184–192) and KFGXGDG (PROSITE acces-sion no. PS00976; aa 407–413) (Fig. 2). One of the putativeNMT sequences, from Brassica oleracea (GenBank Acc. no.AF180355), contained only 1 of these signatures, PS00975(NMT signature 2). This observation casts doubt on its beinga true NMT, since all known NMT amino acid sequencescontain both signatures. This underscores the importance of

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Fig. 1. Phylogenetic analysis of known or putative full-lengthmyristoyl-CoA:protein N-myristoyl transferase (NMTs). NMTamino acid sequences (A) or open reading frame nucleotide se-quences (B) were aligned using clustalw, and the tree was gener-ated using PHYLIP (Felenstein 1989). The trees shown representa consensus of 100 iterations of the neighbor-joining method; allnodes had bootstrap values of at least 80. The scale bars indicatethe branch length corresponding to 0.1 nucleotide or amino acidsubstitutions per site. The sequences used for the alignments aredescribed in Table 2.

experimental validation of putative functional assignmentsthat are commonly based largely on BLAST similarities, es-pecially in large sequencing projects.

Southern blot analysis of various wheat species using theTaNMT probe showed a single hybridizing band correspond-ing to each of the A, B, and D genomes (Fig. 3). This sug-gests that, unlike Arabidopsis (Qi et al. 2000), wheatencodes a single NMT gene and has no NMT2-like se-quence. Consistent with this suggestion is the fact that therice genome contains only 1 gene annotated as NMT-like, al-

though in both wheat and rice an NMT2-like gene may havediverged sufficiently to be undetectable by hybridization orBLAST analysis. Alignment of the rice genomic sequence(GenBank acc. no. AP003273) and the wheat cDNA se-quence showed that the coding region of OsNMT, likeAtNMT (as well as the NMT-like gene in Brassicaoleracea), is likely to be intronless. In Arabidopsis, a single315-bp intron occurs in the 5′ UTR 3 residues upstream ofthe start codon (Qi et al. 2000); however, alignment of the 5′UTR of TaNMT to the genomic region upstream of OsNMT

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Fig. 2. Multiple sequence alignment of known or putative myristoyl-CoA:protein N-myristoyl transferase proteins. The alignment wasgenerated using the clustalw algorithm and displayed using GeneDoc. Conservation of amino acid sequence is represented by shading:black (100%), grey (80%), or white (<60%). The sequences used are described in Table 2.

produced no evidence of an intron in the 5′ UTR of rice(data not shown). The fact that the characterized plant NMTcoding regions are not interrupted by introns whereas animalNMTs commonly contain large introns (Rajala et al. 2000)may reflect the ancient evolutionary split between animalsand plants, suggesting that protein myristoylation evolvedearly in eukaryotic cell evolution. Since expression ofAtNMT1 is observed in a variety of tissues of Arabidopsis(Qi et al. 2000), we analyzed expression of TaNMT by RT-PCR using the primers described; we observed gene expres-sion in all tissues examined (leaf, root, hypocotyl, anther,and ovary; data not shown).

For experimental validation of the identity of TaNMT, weprepared an expression construct in E. coli and characterizedthe recombinant enzyme. As shown in Table 1, recombinantTaNMT behaved as a true NMT, catalyzing the N-myristoylation of peptides derived from the N-terminal se-quences of several putatively N-myristoylated plant proteinsand of known mammalian N-myristoylated proteins. TaNMTdemonstrated an absolute requirement for an N-terminal Gresidue, since the Arabidopsis CDPK6 peptides with an N-terminal A or M residue failed to serve as substrates (Ta-ble 1). Unlike AtNMT (Qi et al. 2000), TaNMT did not dis-play a strong preference for plant-derived peptides,catalyzing the reaction on mammalian A kinase and pp60src-derived peptides with a similar or higher specific activitythan on the plant-derived peptides (Table 1). In addition,there were significant differences in the observed specificactivity with certain peptide substrates between TaNMT andAtNMT; for example, AtNMT had a 4- to 5-fold higher spe-cific activity than TaNMT with Arabidopsis CDPK6 and to-mato Fen kinase, while TaNMT had approximately 2.5-fold

higher specific activity with tobacco CDPK1 (Table 1). Thissuggests that some of the nonconserved residues might beinvolved in peptide substrate recognition. These results con-firm the identity of TaNMT and indicate that the putativelyN-myristoylated proteins from plants that we tested are sub-strates for TaNMT.

In summary, we have shown that a cDNA clone isolatedfrom a leaf, root, and hypocotyl cDNA library of wheat cor-responds to a functional NMT enzyme. This adds a majorphylogenetic group (monocotyledonous plants) to the genesencoding known NMT enzymes and assists in the functionalassignment of several genes (such as OsNMT) that are anno-tated only by BLAST similarities. With the isolation andavailability of TaNMT, it is now possible to address ques-tions related to its biological function in the growth, cell sig-nalling, and development of wheat plants.

Acknowledgements

We acknowledge the use of computing resources providedby the Canadian Bioinformatics Resource (http://cbr-rbc.nrc-cnrc.gc.ca/) and Janet Hill for help in the phylogenetic anal-yses. Frank Vella is thanked for thoughtful critical comments.This is publication number 46593 of the National ResearchCouncil of Canada.

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