Báo cáo khoa học: The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system ppt

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
1
2
3
4
5

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, 3 Bunkyo-cho, Matsuyama,
Ehime 790-8577, Japan
Fax: +81 89 927 8528
Tel: +81 89 927 8274
E-mail:
(Received 10 May 2010, revised 4 July
2010, accepted 8 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 3 do 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, 6 and 7 of the
motif. We found that not only Ser at position 6 but also Lys at position 7
affects the selectivity for the amino acid at position 3. 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 2.

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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, 6 and 7 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, 6 and 7 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 2 (RGLG2) and ADP-ribosylation factor 1
(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

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S. Yamauchi et al.

A

A

B

C

B

Fig. 1. 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. 2. 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 1. 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

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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 3 and 6 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 3 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 3 and 6 for protein N-myristoylation in the

wheat germ cell-free translation system. We generated
cDNAs encoding A. thaliana G protein c subunit 1
(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 3 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 3 differed between Myr–AGG1-3X6A and

Myr–AGG1-3X6S. Only two amino acids (Asn and
Gln) at position 3 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 3 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 3 in protein N-myristoylation, as previously observed in rabbit reticulocyte lysate
[22,34].

A

B
Fig. 3. Effect of Ser6 in the myristoylation
consensus motif on selectivity for the amino
acid at position 3 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

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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 3 (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 1 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 3 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 3 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 3
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 3 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 3 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 3 for protein N-myristoylation shown in Fig. 3.
On the other hand, although proteins in the Met-GlyXaa-Xaa-Xaa-[^Ser]-Lys group do not have Ser6, five
amino acids – Cys, Thr, Asn, Leu, and Gln – were
present at position 3 (Fig. 4C). Therefore, to investigate whether Lys7 also affects the selectivity for amino
acids at position 3 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 3 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 3
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 3 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 3 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 3 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 3 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. 4. Combination of amino acids at positions 3, 6 and 7 in 80 plant proteins with a myristoylation consensus motif. The numbers of the
different amino acids located at position 3 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, 6 and 7 (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 do 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.

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A

Fig. 5. Effects of Ser6 and Lys7 in the
myristoylation consensus motif on selectivity for the amino acid at position 3 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 2. 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 2 (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 3 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 3 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

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S. Yamauchi et al.

A

B

Fig. 6. 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 3 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 do not allow Pro, Asp, Glu, His,
Phe, Lys, Tyr, Trp or Arg at position 3; an arbitrary
amino acid is permitted at positions 4 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 7. 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 3. 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 3
for N-myristoylation. These results indicate that selectivity for the amino acid at position 3 in the plant
myristoylation motif is more complex than that in
S. cerevisiae.
Regarding other amino acid positions, such as positions 4 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, 6 and 7 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

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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 7 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

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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),
5 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 3 h. Samples were denatured by boiling for 3 min 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 min at 4 °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 1 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 min to remove the stain, dehydrated with 100 lL
of 100% acetonitrile for 5 min, and dried for 15 min
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 1 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
described previously [35].

Bioinformatics analysis

Proteins containing objective sequences in the myristoylation
motif were searched for in The Arabidopsis Information
Resource (), with the use of the
pattern matching program ( />patmatch/nph-patmatch.pl). The prediction of protein
N-myristoylation was performed with the plantsp program
( [30].

Acknowledgement
This work was supported by a grant-in-aid for Scientific Research on Priority Areas (Plant Membrane
Transport) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (no.
20053016 to Y. Tozawa).

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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.
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