Unexpected Protein Families Including Cell Defense Components Feature in the N-Myristoylome of a Higher Eukaryote

N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades. We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-myristoylation proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual myristoylation activity. We began with the whole set of N-myristoylated G proteins in the A. thaliana proteome. We then used a reiterative approach, based on the in vitro N-myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1. We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition. The A. thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the N-myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis. The role of N-myristoylation is discussed in each case; in particular, this process may underlie the “guard” hypothesis of innate immunity. N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades. We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-myristoylation proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual myristoylation activity. We began with the whole set of N-myristoylated G proteins in the A. thaliana proteome. We then used a reiterative approach, based on the in vitro N-myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1. We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition. The A. thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the N-myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis. The role of N-myristoylation is discussed in each case; in particular, this process may underlie the “guard” hypothesis of innate immunity. N-terminal methionine excision (NME) 1The abbreviations used are: NME, N-terminal methionine excision; At, A. thaliana; avr, avirulence; F2KP, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase; GP, G protein; R, innate immunity; MYR, N-myristoylation; Myr-CoA, myristoyl-CoA; NMT, myristoyl-CoA:protein N-myristoyltransferase; N-myristoylome, N-myristoylated proteome; NBS-LRR, nucleotide binding site and leucinerich repeat domain(s); ORF, open reading frame; Sc, S. cerevisiae; TF, transcription factor(s); TRX, thioredoxin; Hs, Homo sapiens; PK, protein kinase; ARL, ARF-like protein; ARF, ADP-ribosylation factor; CPK, calcium-dependent protein kinase. is a conserved, essential modification that affects approximately two thirds of the proteins of all proteomes (1Meinnel T. Mechulam Y. Blanquet S. Biochimie (Paris). 1993; 75: 1061-1075Crossref PubMed Scopus (218) Google Scholar, 2Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We use the higher plant Arabidopsis thaliana as a model system for in-depth studies of NME. In A. thaliana, 56% of the proteins of the cytoplasmic proteome are predicted to undergo NME. The molecular basis of NME was recently elucidated in A. thaliana. This pathway involves (i) organellar peptide deformylases (2Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (75) Google Scholar, 4Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (184) Google Scholar), (ii) cytoplasmic and organellar methionine aminopeptidases (2Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 4Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (184) Google Scholar), and (ii) a number of cytoplasmic N-acylases (5Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar, 6Qi Q. Rajala R.V. Anderson W. Jiang C. Rozwadowski K. Selvaraj G. Sharma R. Datla R. J. Biol. Chem. 2000; 275: 9673-9683Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). We need to compare NME substrate characterization data with functional genomics data if we are to understand this process in all the compartments in which it occurs. For example, NME was recently shown to be essential in the plastid, where it stabilizes a small subset of key proteins (8Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (122) Google Scholar). In the cytoplasm, NME leads to a small number of posttranslational N-acylations (9Utsumi T. Sato M. Nakano K. Takemura D. Iwata H. Ishisaka R. J. Biol. Chem. 2001; 276: 10505-10513Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Wilkins M.R. Gasteiger E. Gooley A.A. Herbert B.R. Molloy M.P. Binz P.A. Ou K. Sanchez J.C. Bairoch A. Williams K.L. Hochstrasser D.F. J. Mol. Biol. 1999; 289: 645-657Crossref PubMed Scopus (251) Google Scholar). For example, protein N-terminal myristoylation (MYR) results in the irreversible addition of a saturated C:14 fatty acid to the N-terminal glycines of some proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the transfer of myristate to a number of eukaryotic and viral proteins (for a review on MYR, see Ref. 11Bhatnagar R.S. Ashrafi K. Futterer K. Waksman G. Gordon J.I. Tamanoi F. Sigman D.S. The Enzymes. Vol. XXI. Academic Press, San, Diego2001: 241-289Google Scholar). NMT is essential for viability in protozoans and fungi such as Saccharomyces cerevisiae (12Duronio R.J. Towler D.A. Heuckeroth R.O. Gordon J.I. Science. 1989; 243: 796-800Crossref PubMed Scopus (159) Google Scholar, 13Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar, 14Price H.P. Menon M.R. Panethymitaki C. Goulding D. McKean P.G. Smith D.F. J. Biol. Chem. 2003; 278: 7206-7214Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). It has been suggested that MYR involves important protein components of signal transduction cascades and apoptotic proteins (15de Jonge, H. R., Hogema, B., and Tilly, B. C. (2000) Science's STKE http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2000/63/pe1.Google Scholar). The requirement for the unmasking, by NME, of glycine as the N-terminal residue for the initiation of MYR may account for NME being essential in the cytoplasm of lower and higher eukaryotes. Our studies aim to identify the molecular basis of the requirement for NME in higher eukaryotes. To achieve this goal, we need to characterize all the myristoylated proteins (MYR proteome or N-myristoylome), to identify the most sensitive, primary targets. Recent efforts at N-myristoylome prediction resulted in fine definition of the substrate sequence motif, by reanalysis of comprehensive kinetic and structural data (16Maurer-Stroh S. Eisenhaber B. Eisenhaber F. J. Mol. Biol. 2002; 317: 523-540Crossref PubMed Scopus (158) Google Scholar). This led to the development of a computer program, Predictor, which predicts N-myristoylation by means of two types of analysis, for fungi or higher eukaryotes (17Maurer-Stroh S. Eisenhaber B. Eisenhaber F. J. Mol. Biol. 2002; 317: 541-557Crossref PubMed Scopus (192) Google Scholar). However, regardless of the organism studied, approximately half of all bioinformatic predictions of potential substrates fall into the so-called “twilight zone.” This precludes MYR prediction for these candidates. Therefore, kinetic measurements on N-terminal peptides or full-length proteins are required to obtain definitive data on a given NMT in higher eukaryotes. We recently developed a rapid and reliable MYR diagnostic to achieve this (7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). A straightforward approach to studies of the N-myristoylome is possible as only approximately 0.5% of cytoplasmic proteins are thought to undergo MYR (i.e. approximately 30–200 proteins in a eukaryotic proteome 17). In this study, we aimed to determine the pattern of N-myristoylated proteins in a higher eukaryote proteome (that of A. thaliana) using experimental data obtained directly from the complete proteome. This method was first applied to one class of important signal proteins, GTP-binding proteins (G proteins; GP), and was then extended to several other natural protein substrates for which it was possible to make reliable N-myristoylome predictions. We identified a number of unexpected myristoylated proteins. This study was the first direct analysis of the N-myristoylome of a higher eukaryote. All chemicals were purchased from Sigma. Stock Myr-CoA solutions (200 μm) were dissolved in 1% Triton X-100 in 10 mm sodium acetate buffer, pH 5.6. Oligonucleotides were synthesized at MWG Biotech SA (Courtaboeuf, France), and peptides were synthesized at Eurogentec (Seraing, Belgium). In each case, 2–5 mg of peptide was synthesized, and peptides (85% pure) were dissolved in H2O to give 4 mm solutions. We defined the initiator methionine in polypeptide sequences as Met-1, although this residue is removed by NME. Thus, in peptide sequences, the N-terminal residue, usually a Gly, was numbered as amino acid 2. Nucleotide sequences were determined by the Big-Dye Terminator V3 method, with a 16-capillary ABI Prism 3100 Genetic Analyzer (Applied Biosystems). We obtained pBB131, which encodes Saccharomyces cerevisiae NMT (ScNMT1), from Jeffrey I. Gordon (Washington University, St. Louis, MO). ScNMT1 was excised from this plasmid and inserted into pET16b as previously described (7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). The cDNAs for both the NMT of A. thaliana (AtNMT1 and AtNMT2) were cloned by rapid amplification of cDNA ends from a cDNA library prepared from 2-week-old A. thaliana seedlings as previously described (4Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (184) Google Scholar). The nucleotide sequences of AtNMT1 and AtNMT2 are available from GenBank™, under the accession numbers AF250956 and AF250957, respectively. NMT open reading frames (ORFs) were recloned in pQE31 (Qiagen), to generate N-terminal fusions with a His6 tag, and were then inserted into pET16b. NMT fusions were also inserted between the EcoRI and XhoI restriction sites of the yeast URA3 galactose-inducible plasmid pYES (Invitrogen). The Saccharomyces cerevisiae strains YB332 (MATα ura3 his3Δ200 ade2 lys2–801 leu2) and its thermosensitive derivative YB336 (MATα nmt1–181 ura3 his3Δ200 ade2 lys2–801 leu2) (18Johnson D.R. Duronio R.J. Langner C.A. Rudnick D.A. Gordon J.I. J. Biol. Chem. 1993; 268: 483-494Abstract Full Text PDF PubMed Google Scholar) were provided by J. I. Gordon. YP medium (1% yeast extract, 2% peptone) supplemented with 2% dextrose was used as the standard yeast liquid medium. Yeasts were transformed as described elsewhere (19Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1712) Google Scholar) and cultured at 24 °C. Transformed cells were selected on SD medium (1.8% agar, 1.43 g/liter yeast nitrogen base, 0.5% ammonium sulfate, Clontech yeast dropout amino acid supplement without uracil, 2% dextrose). We checked for effective complementation by culturing cells for 4 days on SD plates supplemented with 2% raffinose and then restreaking them on SD and YP plates supplemented with 2% galactose and incubating them at 24 and 35 °C. Protein production in Escherichia coli was achieved by transforming BL21-pRares (Rosetta; Novagen) cells with a given plasmid construct. Cells were grown at 22 °C for 6 h in 2× TY medium supplemented with 50 μg/ml ampicillin and 34 μg/ml chloramphenicol, to reach an A 600 of 0.9. They were then induced with 0.4 mm isopropyl-1-thio-β-d-galacto-pyranoside and incubated for another 12 h, with shaking, for AtNMT1 and ScNMT1 (7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). For AtNMT2, we used the same conditions, except that the growth medium was supplemented with 3% ethanol and the induction time was 5 h. In all cases, cells were harvested by centrifugation and resuspended in 10–20 ml of buffer A, which consisted of 20 mm sodium phosphate buffer, pH 7.3, 500 mm NaCl, plus 10 mm 2-mercaptoethanol. Samples were subjected to sonication, and cell debris was removed by centrifugation. The supernatant (5–15 ml) was applied to a Hi-Trap chelating HP (0.7 × 2.5 cm; AP-Biotech) nickel affinity column equilibrated in buffer A. Elution was carried out at a flow rate of 0.5 ml/min, in two steps, with buffer B (buffer A plus 0.5 m imidazole) followed by a linear 0.35 mm/min imidazole gradient. The pool of purified protein (5 ml) was first dialyzed against buffer A for 12 h and then against buffer A plus 55% glycerol for 24 h before storage at –20 °C. Matrix-assisted laser desorption ionization time-of-flight analysis (MS Facility, Institut National de la Recherche Agronomique, Jouy, France) of purified AtNMT1 and AtNMT2 indicated that these two proteins were full-length, with molecular masses of 51.3 ± 0.5 and 50.8 ± 0.5 kDa, respectively. These values are entirely consistent with the theoretical values of 50.9 and 50.7 kDa, indicating that the purified enzyme corresponded to the full-length product of the corresponding ORF. Proteins were overproduced in yeast cultured in YP medium containing raffinose (2%) until an A 600 of 0.9 was reached and then induced with 2% galactose. The cells were collected by centrifugation, and the equivalent of 5 A 600 was extracted with glass beads (425–600 μm; Sigma) in a buffer consisting of 50 mm Tris, pH 8.0, 10% glycerol, 5 mm MgCl2, 5 mm EDTA, 1 mm dithiothreitol, and anti-protease mixture (Roche). Protein concentration was determined with the Bio-Rad protein assay kit. Bovine serum albumin was used as the protein standard. Polyacrylamide gel electrophoresis in SDS denaturing gels (10% polyacrylamide gels; 0.75 mm thickness) was performed using the Mini-PROTEAN III system (Bio-Rad). Gels were stained with Bio-Safe Coomassie Stain (Bio-Rad) and blotted onto membranes, which were probed with anti-His antibodies (AP Biotech), as previously described (4Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (184) Google Scholar). NMT activity was assayed at 30 °C by continuously monitoring the absorbance at 340 nm of NADH, by coupling the reaction to pyruvate dehydrogenase activity (7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). The standard assay was performed in a final volume of 200 μl, in 1-cm optical-path quartz cuvettes. Changes in absorbance over time were followed using an Ultrospec-4000 spectro-photometer (AP Biotech), equipped with a temperature control unit and a 6-position Peltier heated cell changer. The reaction mixture contained 50 mm Tris, pH 8.0, 1 mm MgCl2, 0.193 mm EGTA, 0.32 mm dithiothreitol, 0.2 mm thiamine pyrophosphate, 2 mm pyruvate, 0.1 mg/ml bovine serum albumin, 0.1% Triton X-100, 5–1000 μm peptide, 2.5 mm NAD+, and 0.125 unit/ml porcine heart pyruvate dehydrogenase (2 units/mg). A radioactive discontinuous assay (20King M.J. Sharma R.K. Anal. Biochem. 1991; 199: 149-153Crossref PubMed Scopus (73) Google Scholar) was also used to confirm some results (detailed protocol available in Ref. 7Boisson B. Meinnel T. Anal. Biochem. 2003; 322: 116-123Crossref PubMed Scopus (27) Google Scholar). Protein Data—NMT sequences were identified, translated and annotated in several data bases (www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi; www.sanger.ac.uk/DataSearch/omniblast.shtml). Pre liminary sequence data were obtained from (i) the Institute for Genomic Research web site (www.tigr.org) or (ii) the Sanger Institute (www.sanger.ac.uk/). Protein sequences were aligned, using ClustalX software (21Jeanmougin F. Thompson J.D. Gouy M. Higgins D.G. Gibson T.J. Trends Biochem. Sci. 1998; 23: 403-405Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar). The phylogenetic tree was constructed with N-J Tree (21Jeanmougin F. Thompson J.D. Gouy M. Higgins D.G. Gibson T.J. Trends Biochem. Sci. 1998; 23: 403-405Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar) and drawn with TreeView1.65 (taxonomy.zoology.gla.ac.uk/rod/treeview.html; Ref. 22Page R.D.M. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). The original A. thaliana proteome data base (23Arabidopsis Genome InitiativeNature. 2000; 408: 796-815Crossref PubMed Scopus (7211) Google Scholar) was progressively purged of redundant and incorrect entries with the improved annotation developed by the Arabidopsis Genome Initiative (last update February 2003). This was achieved by cross-comparison of the data available at the The Arabidopsis Information Resource (www.arabidopsis.org) and Munich Information Center for Protein Sequences (mips.gsf.de/proj/thal/db/index.html). We extracted ORFs with given patterns from the data base, with the Pattern Matching program (www.arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl; see Ref. 24Cockwell K.Y. Giles I.G. Comput. Appl. Biosci. 1989; 5: 227-232PubMed Google Scholar). The pattern syntax used was: “320<10<0.1GCSVSDKKSOS3-K7D<1>320<10<0.1GCSVSLKKSOS3-K7L>80>320300 ± 304GCSVSAKKSOS3-K7A>90>320100 ± 101.3GCASSLPDRARA6183 ± 1225 ± 47400 ± 400100 (100)GAASSLPDRARA6-C3A286 ± 2427 ± 510600 ± 1200143GCASALPDRARA6-S6A>20>32070 ± 70.9GCASSLADRARA6-P8A300 ± 3042 ± 77300 ± 60098GSSTSGNCBZP44>3>320<10<0.1GNSTSGNCBZP44-S3N400 ± 50325 ± 551200 ± 10016GSSFSGNCBZP44-T5F510 ± 130630 ± 210800 ± 10011a The amino acid sequence of each peptide is indicated. The impact of the position studied is indicated in bold.b See text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.c Values in parentheses refer to data obtained in the radioactive discontinuous assay. Open table in a new tab Table IIThe small G protein N-myristoylome in A. thalianaSequenceaThe amino acid sequence of each peptide is indicated. The impact of the position studied is indicated in bold.EntrybSee text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.NamebSee text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.kcatKmRelative k cat/Km cValues in parentheses refer to data obtained in the radioactive discontinuous assay.× 103 s-1μ m%GCASSLPDRRAt3g54840ARA6 (RABF1)183 ± 1225 ± 4100 (100)GLLCSRSRRAt2g26300GPA1500 ± 10201 ± 534 (85)GLSFAKLFRRAt1g23490ARFA1a166 ± 138 ± 2297 (11)At1g70490ARFA1dAt1g10630ARFA1fGLSFGKLFSRAt2g47170ARFA1c44 ± 31.9 ± 0.5327At3g62290ARFA1eGLNFTKLFRRAt5g14670ARFA1b75 ± 53.5 ± 1.5320GARFSRIAKRAt2g15310ARFB1a100 ± 72.8 ± 0.6497GILFTRMFAt2g24765ARFE67 ± 41.7 ± 0.4185GQTFRKLDTAt5g17060ARFB1b300 ± 2113 ± 3318At3g03120ARFB1cGAFMSRFWRRAt3g22950ARFC1113 ± 71.8 ± 0.4858GTTLGKPFRRAt1g02440ARFD1a155 ± 1112 ± 2189 (80)At1g02430ARFD1bGLLSIIRKAt2g18390ARLC1 (TTN5)>18>3200.9GLLEAFLNAt3g49870ARLA1c<1>320<0.1GLWDSLLNAt5g37680ARLA1a<1>320<0.1At5g67560ARLA1dGAYRAEDDYDAt4g18430RABA1e<1>320<0.1At2g33870RABA1hAt1g28550RABA1iGSSSGQSGRRAt1g43890RABC1>10>3200.4 (1)At5g03530RABC2aAt3g09910RABC2ba The amino acid sequence of each peptide is indicated. The impact of the position studied is indicated in bold.b See text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.c Values in parentheses refer to data obtained in the radioactive discontinuous assay. Open table in a new tab Table IIIKinetic analysis of the MYR of putative substrates of AtNMT1SequenceaThe amino acid sequence of each peptide is indicated. The impact of the position studied is indicated in bold.EntrybSee text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.NamebSee text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.ClassdCB, calcium-binding protein; DE, development; GP, GTP-binding protein; MB, membrane protein; ME, metabolism; PD, protein degradation; PH, phosphatase; PK, protein kinase; R, resistance protein; TF, transcription factor; TR, thioredoxin. Le indicates that the protein sequence was from Lycopersicon esculentum. Ps indicates that the protein sequence was from Pseudomonas syringae. In either case, the two letters are followed by the GenBank™ entry (for instance LeAAF76314). ND, not determined.kcatKmRelative k cat/Km cValues in parentheses refer to data obtained in the radioactive discontinuous assay.×103 s-1μ m%GCASSLPDRRAt3g54840ARA6GP183 ± 1225 ± 4100 (84)GRSSSKKKAt3g59410PK>9>3200.4GDGSSSRSAt5g63610PK>7>3200.3GRAPCCEKRAt3g23250 (+30 others)MYB12TF>2>3200.03 (<0.01)GKKKSDESAt3g54540MB>3>320<0.1GFSFSSNRAt5g17850MB>40>3202GHCYSRNIAt3g49370CRK6PK160 ± 119.3 ± 1.5235GHRHSKSKKKAt4g23650CPK3PKNDNDND (43)eData from Ref. 6.GNANGKDEAt5g21170AKINPK191 ± 30431 ± 956At4g16360GWFKKKSSPsA40613AVRRpt2<1>320<0.1GVKSVRFSAt1g80290>5>3200.1GNACVGPIAt3g10660CPK2PK360 ± 40282 ± 4817GCVLCKESAt5g50860PKPK57 ± 46.5 ± 1.0171GSDGDKKKAt3g06830<1>320<0.1GNVCFRPSPsM22219AVRC188 ± 1520 ± 4130GLLSNRIDAt5g06370<1>320<0.1GSEYKHILAt1g64625PK<1>320<0.1GSRLNFKSRAt4g34000ABF3TF>6>3200.3 (0.3)GGSSGGGVRRAt3g02850SKORMB<1>3200.1 (0.7)GLGASVLTAt5g12860OATMB139 ± 17111 ± 2217GSKYSKATRRLeAAF76314FENPKNDNDND (97)eData from Ref. 6.GCTASKLDRRAt1g02110TF37 ± 35 ± 196GCAQSKIERRAt1g52320TF30 ± 21.5 ± 0.5308GGALSTVFAt5g39950TRXh2TR170 ± 1526 ± 589GLKLSRGPVKAt5g39400PTENPH205 ± 139 ± 2315GSGASKNTRRAt1g07110F2KPME261 ± 1918 ± 2200GQGPSGGLNRAt4g29040RPT2a,bPD350 ± 77900 ± 2005.5At2g20140GAAGSKLEKAAt2g22310UBP3a,bPD93 ± 68 ± 1159At4g39910GASHSHEDRRAt4g33400DEM1DE234 ± 2695 ± 2034(14)GTSQSREDRIAt3g19240DEM2DE160 ± 1111 ± 224GSVMSLGCAt3g44670RPP1R57 ± 44 ± 1185At3g44480At3g44630GISFSIPFAt1g62630NBS-LRR (RPS5-family members)R149 ± 1112 ± 2166At1g63350At1g63360GGCFSVAIAt1g12220RPS5 (and other closely related proteins)R197 ± 1615 ± 4185At1g12210At1g12290At4g14610At5g63020a The amino acid sequence of each peptide is indicated. The impact of the position studied is indicated in bold.b See text for more information. In the AGI annotation, ARA6 corresponds to protein At3g54840, SOS3 to protein At5g24270, and BZP44 to protein At1g75390 in the proteome. Some single substitutions in these three peptides were studied. For instance, in SOS3-G2A, the N-terminal Gly-2 was replaced by an Ala.c Values in parentheses refer to data obtained in the radioactive discontinuous assay.d CB, calcium-binding protein; DE, development; GP, GTP-binding protein; MB, membrane protein; ME, metabolism; PD, protein degradation; PH, phosphatase; PK, protein kinase; R, resistance protein; TF, transcription factor; TR, thioredoxin. Le indicates that the protein sequence was from Lycopersicon esculentum. Ps indicates that the protein sequence was from Pseudomonas syringae. In either case, the two letters are followed by the GenBank™ entry (for instance LeAAF76314). ND, not determined.e Data from Ref. 6Qi Q. Rajala R.V. Anderson W. Jiang C. Rozwadowski K. Selvaraj G. Sharma R. Datla R. J. Biol. Chem. 2000; 275: 9673-9683Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar. Open table in a new tab Enzymatic and Structural Data—The kinetic parameters (k cat, Km , and k cat/Km values) were derived from iterative non-linear least square fits of the Michaelis-Menten equation to the experimental data (25Dardel F. Comput. Appl. Biosci. 1994; 10: 273-275PubMed Google Scholar). Confidence limits for the fitted values were determined by 100 Monte Carlo iterations, using the experimental standard deviations on individual measurements. The amino acid sequence of AtNMT1 was aligned with that of ScNMT1 with InsightII software (Accelrys). The three-dimensional structure of ScNMT1 bound to a peptide and a myristyl-CoA analog (26Farazi T.A. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 6335-6343Crossref PubMed Scopus (65) Google Scholar) was used to construct several three-dimensional models of AtNMT1, with the homology modeler module. The lowest energy structure of AtNMT1 was further minimized with the CharmM forcefield and superimposed on the structure of the peptide bound to ScNMT1. AtNMT1 Complements an S. cerevisiae nmt Conditional Mutation, whereas AtNMT2 Does Not—Thanks to the systematic genome projects underway and the recent release of new data, many putative NMT sequences can now be identified from cDNAs or from genomic fragments. In particular, a number of new plant NMT sequences have been identified. We carried out a phylogenetic analysis of all available full-length putative NMT amino acid sequences (Fig. 1). This analysis indicated that plant NMTs are located between fungal and animal NMTs, on the basis of sequence similarity. Two ORFs encoding two different NMTs were identified in A. thaliana. AtNMT2 was located at the point of divergence from green algae, whereas AtNMT1 displayed strong sequence similarity to NMTs from other dicotyledonou