The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system Seiji Yamauchi1, Naoki Fusada1,2, Hidenori Hayashi1, Toshihiko Utsumi3, Nobuyuki Uozumi4, Yaeta Endo1,5 and Yuzuru Tozawa1 Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Matsuyama, Japan Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa, Japan Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan Systems and Structural Biology Center, RIKEN, Yokohama, Japan Keywords cell-free translation; myristoylation; N-myristoyltransferase; plant; wheat germ Correspondence Y Tozawa, Division of Biomolecular Engineering, Cell-Free Science and Technology Research Center, Ehime University, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan Fax: +81 89 927 8528 Tel: +81 89 927 8274 E-mail: tozaway@ccr.ehime-u.ac.jp (Received 10 May 2010, revised July 2010, accepted July 2010) doi:10.1111/j.1742-4658.2010.07768.x Protein N-myristoylation plays key roles in various cellular functions in eukaryotic organisms To clarify the relationship between the efficiency of protein N-myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell-free translation system with high protein productivity to examine the N-myristoylation of various wild-type and mutant forms of Arabidopsis thaliana proteins Evaluation of the relationship between removal of the initiating Met and subsequent N-myristoylation revealed that constructs containing Pro at position not undergo N-myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase Our analysis of the consensus sequence for N-myristoylation in plants focused on the variability of amino acids at positions 3, and of the motif We found that not only Ser at position but also Lys at position affects the selectivity for the amino acid at position The results of our analyses allowed us to identify several A thaliana proteins as substrates for N-myristoylation that had previously been predicted not to be candidates for such modification with a prediction program We have thus shown that a wheat germ cell-free system is a useful tool for plant N-myristoylome analysis This in vitro approach will facilitate comprehensive determination of N-myristoylated proteins in plants Introduction N-myristoylation is a form of lipid modification that targets a wide variety of eukaryotic proteins and plays important roles in cell physiology In many instances, N-myristoylation alters the lipophilicity of the target protein and facilitates its interaction with membranes, thereby affecting its subcellular localization [1–3] In mammals, N-myristoylated proteins include protein kinases, phosphatases, guanine nucleotide-binding proteins, and Ca2+-binding proteins, many of which participate in signal transduction pathways [1,4–7] Protein N-myristoylation is catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97) (NMT), and involves the covalent attachment of myristic acid, a C14 saturated fatty acid, to the a-amino group of the N-terminal Gly of the target protein This modification usually occurs at the Abbreviations AGG1, Arabidopsis thaliana G protein c subunit 1; ARF1A1c, ADP-ribosylation factor 1; AtNMT1, Arabidopsis thaliana myristoyl-CoA:protein N-myristoyltransferase 1; DHFR, dihydrofolate reductase; GFP, green fluorescent protein; MAP, methionyl aminopeptidase; NMT, myristoyl-CoA:protein N-myristoyltransferase; RGLG2, RING domain ligase 3596 FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS S Yamauchi et al Gly that is exposed during cotranslational elimination of the initiating Met by methionyl aminopeptidase (MAP) [4,8], but the N-terminal Gly revealed after post-translational proteolysis, such as that mediated by caspases, can also be myristoylated if the downstream amino acid sequence matches a consensus motif for myristoylation [9,10] NMT appears to be ubiquitous in eukaryotes, and corresponding genes have been isolated and characterized in many organisms [11–16] In the case of plants, two cDNAs encoding NMT-like proteins, Arabidopsis thaliana NMT1 (AtNMT1) and A thaliana NMT2, have been isolated from A thaliana and characterized, and AtNMT1 has been genetically confirmed to be required for plant viability [17] Most N-myristoylated proteins contain a myristoylation motif, generally defined as Met1-Gly2-Xaa3Xaa4-Xaa5-Ser ⁄ Thr6-Xaa7-Xaa8 (where Xaa indicates any amino acid), at their N-terminus [18] This motif interacts with the substrate-binding pocket of NMT, and thereby ensures binding of the substrate protein to the enzyme [19] Several rules for the myristoylation motif have been proposed, on the basis of biological information such as the N-terminal sequences of known myristoylated proteins and the crystal structures of NMTs [19,20] A biochemical approach showed that the combination of amino acids at positions 3, and in the myristoylation motif is a major determinant of protein N-myristoylation in mammalian systems [21] However, evidence suggests that the consensus sequence for myristoylation in plants differs slightly from those of mammals and yeast [15,17,22] NMT activity has been detected in eukaryotic cellfree translation systems, including rabbit reticulocyte lysate [23], insect [24] and wheat germ extract [25] systems Some of these systems have been applied to the characterization of protein N-myristoylation, and such studies have resulted in the identification of 18 novel human N-myristoylated proteins [26] Wheat germ extracts have proved useful for analysis of N-myristoylation of plant proteins, with N-myristoylation of several A thaliana proteins, such as a Ca2+-dependent protein kinase, a GTPase, and RING finger-type ubiquitin ligases, having been demonstrated in vitro [3,27–29] Two groups have described the ‘N-myristoylome’ of A thaliana [22,30] However, the number of N-myristoylated plant proteins confirmed experimentally has remained far smaller than that predicted in silico, and the consensus motif for plant protein myristoylation is still imprecise We have now clarified the relationship between the efficiency of protein N-myristoylation and the identity of the amino acids at positions 3, and of the consensus motif in plant target proteins with the use of a Cell-free N-myristoylation of plant proteins wheat germ cell-free translation system We identified several new N-myristoylated proteins from A thaliana that were predicted to be noncandidates for N-myristoylation by an existing prediction program Our results thus update the consensus sequence motif for N-myristoylation in plants Results In vitro protein N-myristoylation with a wheat germ cell-free translation system Wheat germ extracts have previously been shown to contain protein N-myristoylation activity [25] To investigate the mode of protein N-myristoylation in plant cells in more detail, we established a cell-free protein N-myristoylation system as an application of an advanced wheat germ cell-free translation system that allows protein synthesis on a preparative scale [31–33] We first analyzed the N-myristoylation of two proteins of A thaliana as model substrates: RING domain ligase (RGLG2) and ADP-ribosylation factor (ARF1A1c) N-myristoylation of these proteins was described previously, and that of RGLG2 was demonstrated in a wheat germ extract [29] We also generated G2A mutants of these proteins as negative controls (nonmyristoylated proteins) by substitution of the codon for Gly2 with a codon for Ala (Fig 1A) The wild-type and mutant proteins were produced with the wheat germ cell-free translation system in the presence of [14C]Leu or [14C]myristic acid, and the translation products were analyzed by SDS ⁄ PAGE and autoradiography In the presence of [14C]Leu, the translation reactions for wild-type and G2A mutants of RGLG2 and ARF1A1c gave rise predominantly to labeled proteins with the expected molecular masses of 52 and 21 kDa, respectively (Fig 1B) In the presence of [14C]myristic acid, however, the wild-type proteins were labeled with 14 C, whereas the G2A mutants were not (Fig 1C) We next analyzed the in vitro N-myristoylation of proteins fused to an N-terminal myristoylation motif For this analysis, we selected green fluorescent protein (GFP) from jellyfish and dihydrofolate reductase (DHFR) from Escherichia coli as model proteins DNA encoding the myristoylation motif Met-Gly-AlaAla-Ala-Ser-Ala-Ala-Ala-Ala was fused to the ORF of GFP or DHFR with the use of PCR to construct genes encoding the chimeric proteins Myr–GFP and Myr–DHFR, respectively (Fig 2A) Furthermore, DNA encoding the N-terminal nonmyristoylation motif Met-Ala-Ala-Ala-Ala-Ser-Ala-Ala-Ala-Ala was also fused to each ORF to construct genes for the G2A mutants Myr–GFP-G2A and Myr–DHFR-G2A FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS 3597 Cell-free N-myristoylation of plant proteins S Yamauchi et al A A B C B Fig In vitro N-myristoylation of two A thaliana substrates (A) Structures and N-terminal amino acid sequences of wild-type (wt) and G2A mutant forms of RGLG2 and ARF1A1c (B, C) The wild-type and G2A mutant proteins were translated in vitro with the use of a wheat germ cell-free translation system in the presence of [14C]Leu (B) or [14C]myristic acid (C) Portions of the reaction products were analyzed by SDS ⁄ PAGE on a 15% gel and autoradiography Control reaction mixtures without mRNA were similarly analyzed Arrowheads indicate proteins of the expected size The positions of protein size standards are shown on the left of the gel (Fig 2A) Translation of mRNAs encoding Myr–GFP or Myr–DHFR in the presence of [14C]Leu gave rise to a single labeled protein with the expected molecular mass of 29 or 25 kDa, respectively (Fig 2B) Such translation in the presence of [14C]myristic acid also showed that Myr–GFP and Myr–DHFR were modified as expected (Fig 2C) By contrast, Myr–GFPG2A and Myr–DHFR-G2A were not labeled with [14C]myristic acid These results thus confirmed that N-myristoylation in the advanced wheat germ cell-free translation system occurs in a manner dependent on the canonical consensus sequence [18] Analysis of in vitro-synthesized N-myristoylated proteins To confirm the N-myristoylation of proteins synthesized with the cell-free system, we next performed MALDI-TOF MS analysis Wild-type forms of ARF1A1c and Myr–GFP were synthesized in vitro and purified by Ni2+-affinity column chromatography on the basis of their His6 tags Tryptic peptides derived 3598 C Fig In vitro N-myristoylation of GFP and DHFR fused to a myristoylation consensus motif (A) Structures and N-terminal amino acid sequences of wild-type (wt) and G2A mutant forms of Myr– GFP and Myr–DHFR (B, C) The wild-type and G2A mutant proteins were translated in vitro with the use of a wheat germ cell-free translation system in the presence of [14C]Leu (B) or [14C]myristic acid (C) Portions of the reaction products were analyzed by SDS ⁄ PAGE on a 15% gel and autoradiography Arrowheads indicate proteins of the expected size from the purified proteins were then analyzed by MALDI-TOF MS Reaction mixtures supplemented with myristic acid yielded a specific peak with m ⁄ z ratios of 818.4 for ARF1A1c and 1184.4 for Myr–GFP (Table 1; Fig S1), values that are in good Table Observed and calculated mass values for the tryptic N-terminal peptides of ARF1A1c and Myr–GFP ND, not detected Observed mass (Da) Sequence of tryptic N-terminal peptide ARF1A1c MGLSFGK GLSFGKa Myristate–GLSFGKb Myr–GFP MGAAASAAAAVSK GAAASAAAAVSKa Myristate–GAAASAAAAVSKb a Calculated mass (Da) Myristic acid (+) Myristic acid ()) 738.9 607.7 818.1 ND ND 818.4 ND 608.4 ND 1105.3 974.1 1184.5 ND ND 1184.4 ND 974.1 ND N-terminal peptide lacking the initiating Met N-terminal peptide b Myristoylated FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS S Yamauchi et al Cell-free N-myristoylation of plant proteins agreement with the calculated mass for the corresponding myristoylated N-terminal fragments (m ⁄ z ratios of 818.1 for ARF1A1c and 1184.5 for Myr–GFP) (Table 1) In the absence of myristic acid, a peak attributable to the N-terminal fragment lacking the initiating Met was detected for both ARF1A1c and Myr– GFP (m ⁄ z ratios of 608.4 and 974.1, respectively) (Table 1; Fig S1) We did not observe a peak corresponding to the N-terminal fragment of either protein that retained the initiating Met in the presence or absence of myristic acid These results thus confirmed removal of the initiating Met and subsequent N-myristoylation of ARF1A1c and Myr–GFP synthesized in the wheat germ cell-free translation system Effect of the combination of amino acids at positions and on protein N-myristoylation in the wheat germ cell-free translation system Ser6 in the myristoylation consensus motif has been shown to affect the selectivity for the amino acid at position in N-myristoylation catalyzed by rabbit reticulocyte lysate [22,34] To clarify further the sequence specificity of the myristoylation motif in plants, we first examined the relationship between amino acids at positions and for protein N-myristoylation in the wheat germ cell-free translation system We generated cDNAs encoding A thaliana G protein c subunit (AGG1) fused at its N-terminus to the myristoylation motifs Met-Gly-Xaa-Ala-Ala-Ala-Ala-Ala-Ala-Ala (Myr–AGG1-3X6A) or Met-Gly-Xaa-Ala-Ala-Ser-AlaAla-Ala-Ala (Myr–AGG1-3X6S), with position of each motif separately occupied by each of the 20 amino acids (Fig 3A) Translation of the corresponding mRNAs in the presence of [14C]Leu gave rise to main products with the expected molecular mass of 13 kDa, indicating that all proteins were effectively translated in the wheat germ system (Fig 3B,C) Translation in the presence of [14C]myristic acid revealed that the requirement of protein N-myristoylation for the amino acid at position differed between Myr–AGG1-3X6A and Myr–AGG1-3X6S Only two amino acids (Asn and Gln) at position allowed efficient N-myristoylation of Myr–AGG1-3X6A (Fig 3B) In contrast, 12 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His, and Met) at position supported efficient N-myristoylation of Myr–AGG1-3X6S (Fig 3C) These results thus indicated that Ser6 in the plant myristoylation motif has a marked effect on selectivity for the amino acid at position in protein N-myristoylation, as previously observed in rabbit reticulocyte lysate [22,34] A B Fig Effect of Ser6 in the myristoylation consensus motif on selectivity for the amino acid at position in initiator Met elimination and N-myristoylation (A) Structure of mature AGG1 fused at its N-terminus to the myristoylation motifs Met-Gly-Xaa-Ala-AlaAla-Ala-Ala-Ala-Ala (Myr–AGG1-3X6A) or Met-Gly-Xaa-Ala-Ala-Ser-Ala-Ala-Ala-Ala (Myr–AGG1-3X6S) (B, C) Each of the 20 mRNAs corresponding to Myr–AGG1-3X6A (B) or Myr–AGG1-3X6S (C) was translated with a wheat germ cell-free translation system in the presence of [14C]Leu (upper panels), [14C]myristic acid (middle panels), or [35S]Met (lower panels) Portions of the reaction products were analyzed by SDS ⁄ PAGE on a 15% gel and autoradiography The molecular masses of labeled translation products are shown on the right C FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS 3599 Cell-free N-myristoylation of plant proteins S Yamauchi et al Relationship between initiator Met elimination and N-myristoylation We next examined the relationship between initiator Met elimination and N-myristoylation with the Myr– AGG1-3X6A and Myr–AGG1-3X6S constructs Given that the mature AGG1 polypeptide does not contain a Met, it was possible to investigate the efficiency of initiator Met elimination by metabolic labeling with [35S]Met In the case of translation products containing Pro at position (Myr–AGG1-3P6A and Myr–AGG1-3P6S), for which the initiating Met is the only Met in the entire encoded amino acid sequence, the extent of [35S]Met incorporation was similar to that observed with Myr– AGG1-3M6A or Myr–AGG1-3M6S (Fig 3B,C), for which Met residues are present at both positions and 3, indicating that the initiating Met is retained in Myr– AGG1-3P6A and Myr–AGG1-3P6S This result is in good agreement with our previous finding of an inhibitory effect of Pro at position of full-length translation products on MAP function in the wheat germ cell-free translation system [35] We also detected low levels of [35S]Met incorporation in the translation products with Gly, Thr, Asp or Glu at position of the myristoylation motif in Myr–AGG1-3X6A or Myr–AGG1-3X6S (Fig 3B,C) Given that the AGG1 mutants containing Pro3 were not modified by N-myristoylation (Fig 3B,C), our results indicate that Pro at position prevents substrate recognition not by NMT but rather by MAP, which functions upstream of NMT Effect of Lys7 in the myristoylation motif on selectivity for the amino acid at position in protein N-myristoylation Podell and Gribskov recently developed a program (plantsp) for the prediction of N-myristoylation sites in plant proteins [30] The construction of this program relied on 80 plant proteins selected on the basis of direct evidence for their N-myristoylation, subcellular localization, and N-terminal sequence conservation We examined the N-terminal sequences of these 80 proteins, and categorized them into four groups: 18 proteins (22.5%) without Ser6 and Lys7, designated the Met-Gly-Xaa-Xaa-Xaa-[^Ser]-[^Lys] group (where [^Ser] and [^Lys] mean that Ser and Lys are excluded); 26 proteins (32.5%) with Ser6 but without Lys7, designated the Met-Gly-Xaa-Xaa-Xaa-Ser-[^Lys] group; 14 proteins (17.5%) without Ser6 but with Lys7, designated the Met-Gly-Xaa-Xaa-Xaa-[^Ser]-Lys group; and 22 proteins (27.5%) with both Ser6 and Lys7, designated the Met-Gly-Xaa-Xaa-Xaa-Ser-Lys group The numbers of individual amino acids located at 3600 position in each group were then counted (Fig 4) In the Met-Gly-Xaa-Xaa-Xaa-[^Ser]-[^Lys] group, most proteins (17 of 18) had Asn3 (Fig 4A), whereas various amino acids were present at this position in the Met-Gly-Xaa-Xaa-Xaa-Ser-[^Lys] and Met-Gly-XaaXaa-Xaa-Ser-Lys groups (Fig 4B,D) These results are consistent with the amino acid requirements at position for protein N-myristoylation shown in Fig On the other hand, although proteins in the Met-GlyXaa-Xaa-Xaa-[^Ser]-Lys group not have Ser6, five amino acids – Cys, Thr, Asn, Leu, and Gln – were present at position (Fig 4C) Therefore, to investigate whether Lys7 also affects the selectivity for amino acids at position in the plant myristoylation motif, we constructed cDNAs encoding Myr–AGG13X6A7K and Myr–AGG1-3X6S7K, corresponding to Myr–AGG1-3X6A and Myr–AGG1-3X6S, respectively, with Ala7 changed to Lys (Fig 5A) We then examined these constructs for initiator Met elimination and N-myristoylation by metabolic labeling All of the constructs were efficiently translated as determined from the incorporation of [14C]Leu (Fig 5B,C), and the pattern of [35S]Met incorporation was the same as that for the corresponding Myr–AGG1-3X6A and Myr–AGG1-3X6S constructs (data not shown) Labeling with [14C]myristic acid revealed that the selectivity for amino acids at position for N-myristoylation in the Myr–AGG1-3X6A7K constructs was the same as that observed with Myr–AGG1-3X6S; that is, 12 amino acids – Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His, and Met – were permitted at position for N-myristoylation (Fig 5B) In the case of Myr– AGG1-3X6S7K, [14C]myristic acid incorporation was detected in all constructs, with the exception of those with Pro3 or Asn3 (Fig 5C), indicating that selectivity for the amino acid at position for N-myristoylation in Myr–AGG1-3X6S7K was extended relative to that in Myr–AGG1-3X6S or Myr–AGG1-3X6A7K Together, these results indicated that not only Ser6 but also Lys7 contributes to selectivity for the amino acid at position for protein N-myristoylation in plants The combination of Ser6 and Lys7 in the motif thus allows more varieties of acceptable amino acids at position than that of Ser6 and [^Lys]7 This result updated the consensus sequence for N-myristoylation In vitro N-myristoylation of A thaliana proteins predicted not to be candidates for the modification To perform an N-myristoylation assay based on the results shown in Figs and 5, we selected eight genes from a search of potential N-myristoylated proteins in FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS S Yamauchi et al Cell-free N-myristoylation of plant proteins A B C D Fig Combination of amino acids at positions 3, and in 80 plant proteins with a myristoylation consensus motif The numbers of the different amino acids located at position in the 80 plant proteins with a myristoylation consensus motif listed in a recent study [30] were counted Results are shown for proteins in the Met-Gly-Xaa-Xaa-Xaa-[^Ser]-[^Lys] group (A), Met-Gly-Xaa-Xaa-Xaa-Ser-[^Lys] group (B), MetGly-Xaa-Xaa-Xaa-[^Ser]-Lys group (C), or Met-Gly-Xaa-Xaa-Xaa-Ser-Lys group (D) the A thaliana database All of these proteins were predicted not to be candidates for N-myristoylation with the plantsp program, but they possess an amino acid sequence consistent with the new variation of the myristoylation consensus motif identified in the present study and based on the combination of amino acids at positions 3, and (Table 2) We analyzed the eight proteins for potential N-myristoylation with the wheat germ cell-free translation system In the presence of [14C]Leu, the translation products of At1G64850, At4G00305, At3G55450, At5G03200 and At5G03870 were detected at positions corresponding to their expected molecular masses (18, 14, 43, 38 and 43 kDa, respectively), whereas those of At1G66480, At3G18430 and At5G64690 were detected at positions corresponding to molecular masses larger than those calculated (25, 20 and 38 kDa, respectively) (Fig 6A) In all instances, proteins labeled with [14C]myristic acid were detected at positions similar to those of the proteins labeled with [14C]Leu (Fig 6B), indicating that all eight proteins were N-myristoylated With the use of cell-free analysis, we were thus able to identify novel substrates for N-myristoylation that had not previously been shown to undergo such modification by biochemical analysis and were not predicted to so with the plantsp program Discussion Protein N-myristoylation promotes the membrane association that is essential for appropriate protein localization, and N-myristoylated proteins play key roles in various cellular functions [1,4,8] We have applied an advanced wheat germ cell-free translation system as a tool to characterize protein N-myristoylation in plants We first evaluated the utility of this system for analysis of N-myristoylated proteins N-myristoylation of the tested proteins was detected by labeling with [14C]Leu and [14C]myristic acid, and was confirmed by MS analysis FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS 3601 Cell-free N-myristoylation of plant proteins S Yamauchi et al A Fig Effects of Ser6 and Lys7 in the myristoylation consensus motif on selectivity for the amino acid at position in N-myristoylation (A) Structure of mature AGG1 fused at its N-terminus to the myristoylation motifs Met-Gly-Xaa-Ala-Ala-Ala-Lys-Ala-AlaAla (Myr–AGG1-3X6A7K) or Met-Gly-Xaa-AlaAla-Ser-Lys-Ala-Ala-Ala (Myr–AGG1-3X6S7K) (B, C) Each of the 20 mRNAs corresponding to Myr–AGG1-3X6A7K or Myr–AGG13X6S7K was translated with the use of a wheat germ cell-free translation system in the presence of [14C]Leu (upper panels) or [14C]myristic acid (lower panels) Portions of the reaction products were analyzed by SDS ⁄ PAGE on a 15% gel and autoradiography The molecular masses of labeled translation products are shown on the right B C Table Selected A thaliana proteins for analysis of N-myristoylation in vitro N-myristoylation prediction was performed with the N-myristoylation prediction program [30] PLANTSP A thaliana database entry GenBank accession number N-terminal sequence N-myristoylation prediction Prediction score Product At1G64850 At1G66480 At3G18430 At3G55450 At4G00305 At5G03200 At5G03870 At5G64690 NM_105159 NM_105319 NM_112728 NM_115403 NM_116252 NM_120398 NM_120468 NM_125865 MGQVFNKLRG MGNSITVKRK MGNTSSMLTQ MGSCLSSRVL MGLSYSGAGV MGNLISLIFC MGCVSSKLGK MGNCAIKPKV – – – – – – – – )3.7 )1.6 )2.6 )1.8 )2.2 )2.3 )0.9 0.3 Ca2+-binding EF-hand family protein Plastid movement impaired (PMI2) Ca2+-binding EF-hand family protein Protein kinase Zinc-finger (C3HC4-type RING finger) family protein Zinc-finger (C3HC4-type RING finger) family protein Glutaredoxin family protein Neurofilament triplet H protein Given that myristic acid is attached to a Gly at the N-terminus of a protein, the initiating Met must be cleaved by MAP prior to N-myristoylation We have previously shown that a Pro at position markedly inhibits cleavage of the initiator Met by MAP, even if the penultimate amino acid is Ala, Cys, Gly, Pro, Ser, or Thr, all of which generally allow efficient Met removal from a translated polypeptide [35] Before this finding, the antepenultimate amino acid had not been thought to affect the substrate selectivity of MAP Rather, a Pro at position had been considered to affect substrate recognition by NMT [34] In the present study, by taking advantage of the fact that the mature AGG1 polypeptide does not contain Met, we also examined the efficiency of elimination of the 3602 initiator Met in a series of mutants by labeling with [35S]Met We found that the translation products of AGG1 mutants containing Pro3 retained [35S]Met, indicating that cleavage of the initiating Met by MAP did not occur This result is thus consistent with our previous data obtained from analysis of the sequence specificity and efficiency of endogenous MAP activity in the wheat germ cell-free translation system [35] The AGG1 mutant proteins containing Pro3 were not modified by N-myristoylation We thus demonstrated that constructs containing the amino acid sequence MetGly-Pro at their N-terminus are not myristoylated, primarily because of the substrate specificity of MAP With respect to the consensus motif for N-myristoylation, although Gly2 is absolutely required for protein FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS S Yamauchi et al A B Fig In vitro N-myristoylation of A thaliana proteins predicted not to be substrates for the modification Eight proteins, all of which were predicted to be negative candidates for N-myristoylation by the PLANTSP program, were translated in vitro with a wheat germ cell-free translation system in the presence of [14C]Leu (A) or [14C]myristic acid (B) Portions of the reaction products were analyzed by SDS ⁄ PAGE and autoradiography Open and closed arrowheads indicate the positions of full-length proteins and products of aborted translation, respectively N-myristoylation, not all proteins with Gly2 are N-myristoylated Previous studies have described preferences for certain amino acids at distinct positions downstream of the N-terminal Gly Mammalian cellfree translation systems have shown that Ser6 greatly affects the selectivity for amino acids at position in protein N-myristoylation [21,34] In addition, a set of empirical rules for a myristoylation motif has been proposed on the basis of the structure of Saccharomyces cerevisiae NMT and of in vitro kinetic analysis of the purified protein and synthetic peptide substrates [19,36] These rules not allow Pro, Asp, Glu, His, Phe, Lys, Tyr, Trp or Arg at position 3; an arbitrary amino acid is permitted at positions and 5; only Ser, Thr, Ala, Gly, Cys and Asn are permitted at position 6; and only Pro among the 20 amino acids is not allowed at position By evaluating N-myristoylation efficiency for a series of AGG1 mutants with a fused myristoylation motif, we have now shown that not only Ser6 but also Lys7 influences selectivity of N-myristoylation for the amino acid at position Furthermore, in the case of the constructs containing Ser6 and Cell-free N-myristoylation of plant proteins Lys7, only Pro and Asn were not allowed at position for N-myristoylation These results indicate that selectivity for the amino acid at position in the plant myristoylation motif is more complex than that in S cerevisiae Regarding other amino acid positions, such as positions and 5, we have not yet investigated the precise selectivity of amino acids in the plant N-myristoylation system Investigations of these positions could further update the consensus sequence A prediction program for N-myristoylation of plant proteins, plantsp, was recently developed [30] However, given that only a small number of N-myristoylated proteins have been experimentally confirmed in plants, the accuracy of N-myristoylation prediction has not yet achieved sufficient reliability Indeed, our in vitro analysis of AGG1 mutants revealed that the prediction program was not sufficiently effective for evaluation of N-terminal amino acid sequences as potential N-myristoylation motifs (Table S1) For example, although effective N-myristoylation was observed in Myr–AGG1-3X6S constructs with Gly, Ala, Ser, Thr, Val, Leu, Ile, Gln, His or Met at position 3, the prediction program gave low scores for the possibility of N-myristoylation of these constructs (Table S1) We further selected eight A thaliana proteins on the basis of combinations of amino acids at positions 3, and that our results suggested would be compatible with N-myristoylation, and we examined whether this was the case with the cell-free system Although these proteins were predicted to be negative candidates by the plantsp program, we detected their myristoylation in vitro (Fig 6) To date, 319 proteins of A thaliana, representing 1.1% of the total proteome, have been predicted to be N-myristoylation candidates [30] According to the results obtained from our experiments, we further surveyed A thaliana proteins that are potentially N-myristoylated Using the pattern matching program in The Arabidopsis Information Resource, we listed 103 A thaliana proteins (Table S2) Among these, 79 were included in the above-mentioned 319 proteins, but the other 24 were newly found candidates These results suggest that the actual number of N-myristoylated proteins in A thaliana may be substantially larger than the number previously predicted Our investigation is based on wheat germ extracts We therefore need to note that the substrate specificity of the wheat NMT could be technically different from that of the A thaliana enzyme On the other hand, a single-copy gene encoding an AtNMT1 homolog (73% identity) has been reported previously as a candidate gene for wheat NMT [16] We suppose that the wheat FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS 3603 Cell-free N-myristoylation of plant proteins S Yamauchi et al homolog of AtNMT1 is most likely the enzyme responsible for the protein N-myristoylation occurring in the wheat germ extracts The wheat germ cell-free N-myristoylation system is thus useful for the detection and characterization of N-myristoylated proteins in plants The substrate specificity for N-myristoylation of plant proteins revealed by the wheat germ cell-free myristoylation system will facilitate the preparation of N-myristoylated proteins at a preparative scale, as well as the high-throughput and comprehensive proteomic analysis of N-myristoylated plant proteins Experimental procedures Plasmid construction All restriction endonucleases and DNA-modifying enzymes for plasmid construction were obtained from Takara Shuzo (Kyoto, Japan) We modified the pEU3S cell-free expression vector [37] by introducing a nucleotide sequence into the 3¢-end of the multiple cloning site, so that the C-terminus of the encoded polypeptide would be conjugated to a His6 tag To prepare a DNA fragment encompassing the modified multiple cloning site, we performed PCR with KOD plus DNA polymerase (Toyobo, Osaka, Japan), primers pEU3S FW and pEU3S RV (Table S3), and the pEU3S vector as a template The amplified DNA fragment was digested with SpeI and NcoI, and then cloned into the corresponding SpeI–NcoI sites of pEU3S The resulting plasmid was designated pEU3SH The coding regions of RGLG2 (GenBank accession no NM_203051) and ARF1A1c (NM_130285) of A thaliana were obtained by RT-PCR with Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), KOD plus DNA polymerase (Toyobo), and corresponding PCR primers (Table S3) The PCR product for RGLG2 was digested with SpeI and SmaI, and that for ARF1A1c was digested with XhoI and SmaI The resultant DNA fragments were then cloned into the corresponding sites of pEU3SH For expression of N-myristoylated GFP and E coli DHFR, each coding region fused with a DNA sequence encoding the myristoylation motif Met-Gly-Ala-Ala-Ala-Ser-Ala-AlaAla-Ala was amplified by PCR with appropriate primers (Table S3), digested with SpeI and SmaI, and cloned into the corresponding sites of pEU3SH Each G2A construct was prepared by PCR with corresponding G2A primers (Table S3) and the corresponding pEU3SH-based vector as the template, and was then cloned into pEU3SH as described above For further analysis of N-myristoylation of A thaliana proteins, the coding regions of At1G64850 (NM_105159), At1G66480 (NM_105319), At3G18430 (NM_112728), At3G55450 (NM_115403), At4G00305 (NM_116252), 3604 At5G03200 (NM_120398), At5G03870 (NM_120468) and At5G64690 (NM_125865) were obtained by RT-PCR as described above and with the primers listed in Table S3 The PCR products of At1G66480, At3G18430 and At5G03870 were digested with SpeI and BamHI, and the other PCR products were digested with SpeI and BglII The resultant DNA fragments were then cloned into the SpeI and BglII sites of the pEU3b vector [37] For analysis of the sequence specificity of the myristoylation motif, the coding region of A thaliana AGG1 (NM_116207) was obtained by RT-PCR as described above and with appropriate primers (Table S3) The PCR product was cloned into the pTA2 vector with the use of TArget Clone (Toyobo), yielding pTA2–AGG1 The coding region for mature AGG1 fused with a DNA fragment encoding the myristoylation motif Met-Gly-Ala-Ala-Ala-Ala-Ala-AlaAla-Ala or Met-Gly-Ala-Ala-Ala-Ser-Ala-Ala-Ala-Ala was amplified by PCR with the primers AGG1 RV and 3A6(A ⁄ S) FW (Table S3) and with pTA2–AGG1 as the template The resultant DNA fragments were cloned into the SpeI and SmaI sites of pEU3SH for production of Myr– AGG1-3A6A or Myr–AGG1-3A6S, respectively The cDNAs encoding Myr–AGG1-3X6A and Myr–AGG13X6S, corresponding to Myr–AGG1-3A6A or Myr–AGG13A6S, respectively, with Ala3 replaced with each of the other 19 amino acids, were constructed by PCR with the primer pEU3SH RV and the corresponding 3X6(A ⁄ S) FW primer (Table S3) and with the Myr–AGG1-3A6A and Myr– AGG1-3A6S vectors as templates The resultant DNA fragments were digested with SpeI and BglII, and then cloned into the corresponding sites of pEU3b For further substitution of Lys for Ala at position of Myr–AGG1-3X6A, the cDNA encoding Myr-AGG1-3A6A7K was constructed by PCR with the primers pEU3SH RV and 3A6A7K FW (Table S3) and with the Myr–AGG1-3A6A vector as template The PCR product was cloned into pEU3b as described above The cDNAs encoding Myr–AGG1-3X6A7K were constructed by an approach similar to that used for construction of Myr–AGG1-3X6A cDNAs, with the primers listed in Table S3 and with the Myr–AGG1-3A6A7K vector as the template The cDNAs encoding Myr–AGG13X6S7K, corresponding to Myr–AGG1-3X6A7K with Ser substituted for Ala6, were constructed as described above with appropriate primers (Table S3) and with the corresponding Myr–AGG1-3X6A7K vector as template The sequences of all constructs were confirmed by DNA sequencing with the use of an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) In vitro N-myristoylation assay For detection of N-myristoylation of A thaliana proteins, the corresponding plasmid (10 lg) purified with the use of a Qiagen Midi Kit (Qiagen, Chatsworth, CA, USA) was used as a template for in vitro transcription with SP6 RNA FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS S Yamauchi et al polymerase (Promega, Madison, WI, USA) In the case of analysis of the sequence specificity of the myristoylation motif, template DNAs for in vitro transcription were prepared from the Myr–AGG1-3X6A, Myr–AGG1-3X6S, Myr–AGG1-3X6A7K and Myr–AGG1-3X6S7K vectors by PCR Proteins were synthesized by the batch method, with the use of a wheat germ cell-free translation system (Wepro; CellFree Sciences, Matsuyama, Japan) in the presence of [14C]Leu (Perkin Elmer, Waltham, MA, USA), [14C]myristic acid (American Radiolabeled Chemicals, St Louis, MO, USA), or [35S]Met (American Radiolabeled Chemicals), under the conditions recommended by the manufacturer [38] The batchwise reaction mixture (25 lL) contained wheat germ extract (final concentration, 60 A260 nm units), 4.7 lL of reaction buffer, creatine kinase (0.4 mgỈmL)1), lL of mRNA, and either 0.5 lL of [14C]Leu (316 CiỈmol)1, 100 lCiỈmL)1), 0.5 lL of [14C]myristic acid (55 CiỈmol)1, 100 lCiỈmL)1), or 0.25 lL of [35S]Met (810.3 CiỈmmol)1, 10.89 mCiỈmL)1), and was incubated at 26 °C for h Samples were denatured by boiling for in SDS sample buffer, and were then analyzed by SDS ⁄ PAGE on a 15% gel The gel was dried under vacuum and then subjected to autoradiography Protein purification Myristoylated or nonmyristoylated proteins were synthesized with the cell-free translation system with or without the addition of myristic acid (Nacalai tesque, Kyoto, Japan) at a final concentration of 75 lm The reaction mixture was centrifuged at 20 400 g for 20 at °C, and the resulting supernatant was mixed with 10 volumes of a solution containing 50 mm Tris ⁄ HCl (pH 7.5), 500 mm NaCl, and 20 mm imidazole (buffer A) and then applied to a mL HiTrap chelating column (GE Healthcare, Little Chalfont, UK) that had been equilibrated with buffer A The column was washed with 10 mL of buffer A containing 40 mm instead of 20 mm imidazole, and then with 10 mL of buffer B (50 mm Tris ⁄ HCl, pH 7.5, 50 mm NaCl, 40 mm imidazole) Elution was then performed with buffer B containing 400 mm instead of 40 mm imidazole, as well as 10% glycerol, and the eluate was stored at )80 °C until use MALDI-TOF MS analysis Purified N-myristoylated or nonmyristoylated proteins were separated by SDS ⁄ PAGE on a 12.5% gel and stained with Coomassie Brilliant Blue R250 The stained protein bands were excised from the gel, incubated in 100 lL of 30% acetonitrile containing 25 mm ammonium bicarbonate for 10 to remove the stain, dehydrated with 100 lL of 100% acetonitrile for min, and dried for 15 under vacuum The protein bands were then reduced and alkylated before digestion and extraction with the use of an XL-trypKit (Promega) The extracted peptides were desalt- Cell-free N-myristoylation of plant proteins ed and concentrated with the use of a ZipTip C18 device (Millipore, Billerica, MA, USA), and samples eluted from the device with lL of saturated a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St Louis, MO, USA) in 90% acetonitrile containing 0.1% trifluoroacetic acid were spotted onto a MALDI target plate (Applied Biosystems) MALDI-TOF mass spectra were acquired with a Voyager DE Biospectrometry Workstation (Applied Biosystems) as 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enzyme in the plant tryptophan pathway Plant Physiol 138, 2260–2268 38 Sawasaki T, Ogasawara T, Morishita R & Endo Y (2002) A cell-free protein synthesis system for highthroughput proteomics Proc Natl Acad Sci USA 99, 14652–14657 Cell-free N-myristoylation of plant proteins Supporting information The following supplementary material is available: Fig S1 MALDI-TOF mass spectra of tryptic digests derived from in vitro-synthesized ARF1A1c or Myr– GFP Table S1 Summary of the results of N-myristoylation analysis and prediction for four series of AGG1 mutants Table S2 List of predicted proteins containing an N-myristoylation motif at the N-terminus in A thaliana Table S3 Oligonucleotide primers used in the study This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3596–3607 ª 2010 The Authors Journal compilation ª 2010 FEBS 3607 ... myristoylation motif Met-Gly-Ala-Ala-Ala-Ala-Ala-AlaAla-Ala or Met-Gly-Ala-Ala-Ala-Ser-Ala-Ala-Ala-Ala was amplified by PCR with the primers AGG1 RV and 3A6 (A ⁄ S) FW (Table S3) and with pTA2–AGG1 as the. .. to the myristoylation motifs Met-Gly-Xaa-Ala-AlaAla-Ala-Ala-Ala-Ala (Myr–AGG1-3X 6A) or Met-Gly-Xaa-Ala-Ala-Ser-Ala-Ala-Ala-Ala (Myr–AGG1-3X6S) (B, C) Each of the 20 mRNAs corresponding to Myr–AGG1-3X 6A. .. N-terminus to the myristoylation motifs Met-Gly-Xaa-Ala-Ala-Ala-Ala-Ala-Ala-Ala (Myr–AGG1-3X 6A) or Met-Gly-Xaa-Ala-Ala-Ser-AlaAla-Ala-Ala (Myr–AGG1-3X6S), with position of each motif separately occupied