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INTRODUCTION |
A number of eukaryotic cellular proteins are found to be
covalently modified with the 14-carbon saturated fatty acid, myristic acid (1-6). Many of the myristoylated proteins play key roles in
regulating cellular structure and function. Protein
N-myristoylation is the result of the cotranslational
addition of myristic acid to a Gly residue at the extreme N terminus
after removal of the initiating Met. The requirement for Gly at the N
terminus is absolute, and no other amino acid can take its place. A
stable amide bond links myristic acid irreversibly to proteins. The
N-myristoyltransferase (NMT),1 which catalyzes the
transfer of myristic acid from myristoyl-CoA to the N-terminal Gly has
been purified and cloned from several organisms (7-10). The precise
substrate specificity of this enzyme has been characterized using
purified enzyme and synthetic peptide substrates (2, 11, 12). In
general, Ser or Thr is preferred at position 6, and the N-terminal
consensus motifs such as
Met-Gly-X-X-X-Ser/Thr-X-X (13) or
Met-Gly-X-X-X-Ser/Thr-X-X-X
(5) that direct protein N-myristoylation have been defined.
However, Ser or Thr at position 6 is neither sufficient nor critical
for the recognition of the protein substrate by the NMT. For instance,
the peptide Gly-Gln-Ala-Ala-Ala-Ala-Lys-Lys derived from the N terminus
of the cAMP-dependent protein kinase catalytic subunit was
found to be a good substrate for the yeast NMT, and the peptide
Gly-Gln-Ala-Ala-Ala-Ala-Arg-Arg was used as a reference substrate for
the yeast NMT in earlier reports on the substrate specificity of this
enzyme (7, 14). Some amino acid preferences were also reported at other
positions such as 3, 7, and 8 (2, 15); however, the precise amino acid
requirements at these positions were not fully characterized.
Protein N-myristoylation in intact cells is not a single
enzymatic reaction catalyzed by NMT. This modification appears to be a
highly regulated reaction involving the coordinated participation of
the protein synthesis machinery (ribosomes) and several different enzymes/proteins such as N-methionylaminopeptidase, fatty
acid synthase, long chain acyl-CoA synthetase, acyl-CoA-binding
proteins, etc. Therefore, the substrate specificity of NMT analyzed by
using purified NMT and synthetic peptide substrates may not fully
reflect the substrate specificity of NMT in intact cells. In addition, other cotranslational protein modification such as protein
N-acetylation might also affect the reaction. It has been
estimated that as many as 70% of soluble proteins (cytoplasmic or
nucleoplasmic) in eukaryotes bear this modification (16). In fact,
several proteins having an N-terminal Gly residue, such as ovalbumin
(17), cytochrome c (18), and actin (19), were found to be
N-acetylated. However, the difference in the N-terminal
sequence requirement for protein N-myristoylation and
protein N-acetylation has not been characterized so far.
Since the in vitro translation system using rabbit
reticulocyte lysate contains the components involved in cotranslational protein N-myristoylation and N-acetylation (17,
19, 20), the use of this system to study cotranslational protein
modification seems to be appropriate. In fact, we previously
demonstrated that tumor necrosis factor (TNF), a nonmyristoylated model
protein, could be efficiently myristoylated in the in vitro
translation system when an N-myristoylation motif of Rasheed
leukemia virus-Gag protein or Gi1
protein was
linked to the mature domain of TNF (21, 22).
In this study, to examine the N-terminal sequence requirements for the
cotranslational protein N-myristoylation and to reveal the
difference in the N-terminal sequence requirements for protein N-myristoylation and N-acetylation, several
series of site-directed mutagenesis of the N-terminal region of protein
were performed using TNF as a nonmyristoylated model protein.
Subsequently, the susceptibility of these mutants to the
cotranslational N-myristoylation and
N-acetylation reactions was evaluated by an in
vitro transcription/translation system using the rabbit
reticulocyte lysate.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases, DNA-modifying enzymes,
RNase inhibitor, and Taq DNA polymerase were purchased from
Takara Shuzo (Kyoto, Japan). The mCAP RNA capping kit and
proteinase K were from Stratagene. RNase was purchased from Roche
Molecular Biochemicals. Rabbit reticulocyte lysate was from Promega.
[3H]leucine, [3H]myristic acid,
[3H]acetyl-CoA, [35S]methionine, and
Amplify were from Amersham Pharmacia Biotech. The Dye Terminator Cycle
Sequencing kit was from Applied Biosystems. Anti-human TNF polyclonal
antibody was purchased from R & D Systems. Protein G-Sepharose was
from Amersham Pharmacia Biotech. Plasmid pET-22b-OVA, which contains
the full-length chicken ovalbumin cDNA, was provided by Dr. Akio
Kato (Yamaguchi University, Japan). Other reagents purchased from Wako
Pure Chemical, Daiichi Pure Chemicals, and Seikagaku Kogyo (Japan) were
of analytical or DNA grade.
Plasmid Construction--
Plasmid pBluescript II SK(+) lacking
ApaI and HindIII sites was constructed as
previously described (23) and designated pB. Plasmid pBpro-TNF, which
contains the full-length human pro-TNF cDNA, and plasmid
pB
pro-TNF, containing a cDNA coding for the mature domain of
TNF, were constructed as described (21, 23). Plasmid pBV2G-TNF was
constructed by utilizing PCR. For this procedure, pB
pro-TNF served
as a template, and two oligonucleotides (V2G and B1) served as primers
(Table I).
After digestion with BamHI and PstI, the
amplified product was subcloned into pB at BamHI and
PstI sites. The cDNAs coding for other TNF mutants
(designated R3X-TNF), in which Arg at position 3 in V2G-TNF was
replaced with each of the 19 other amino acids, were constructed by a
method similar to that of V2G-TNF. The mutagenic primers used in these
procedures are listed in Table I. Plasmids pBGag-TNF and
pBGi1
-TNF were constructed as described previously (22).
Plasmid pBOVA, which contains the full-length chicken ovalbumin
cDNA was constructed by using PCR. In this case, pET-22b-OVA served
as a template, and two oligonucleotides (OVA-N and OVA-C) served as
primers (Table I). After digestion with BamHI and
EcoRI, the amplified product was subcloned into pB at
BamHI and EcoRI sites. The cDNA coding for
OVA60-TNF in which the N-terminal 60 residues of ovalbumin were linked
to the N terminus of the mature domain of TNF was constructed by using
PCR. For this procedure, pBOVA served as a template, and two
oligonucleotides (OVA-N and OVA-60) served as primers (Table I). After
digestion with BamHI and XhoI, the amplified
product was subcloned into pBGag-TNF at BamHI and
XhoI sites. The cDNAs coding for R3X,S6A-TNF in which Ser at position 6 in R3X-TNF mutants was replaced with Ala, were constructed by using PCR. In this case, each of the pBR3X-TNF constructs served as a template, and two oligonucleotides (T3 and S6A)
served as primers (Table I). After digestion with SacI and
AvaI, the amplified product was subcloned into pB
pro-TNF at SacI and AvaI sites. The cDNAs coding for
Gag-Q3K-TNF and Gi1
-C3K-TNF in which the amino acid at
position 3 in Gag-TNF or Gi1
-TNF was replaced with Lys
were constructed by using PCR. In this case, pBGag-TNF or
pBGi1
-TNF served as a template, and two oligonucleotides (Gag-Q3K plus B1 and Gi1-C3K plus B1, respectively) as primers (Table I). After digestion with BamHI and PstI,
the amplified products were subcloned into pB at BamHI and
PstI sites. The cDNAs coding for Arf6-TNF and
hippocalcin-TNF, in which the N-terminal 10 residues of
pro-TNF were
replaced with those of Arf6 or hippocalcin, were constructed by using
PCR. For this procedure, pB
pro-TNF served as a template, and two
oligonucleotides (Arf6 plus B1 and HC plus B1, respectively)
served as primers (Table I). After digestion with BamHI and
PstI, the amplified products were subcloned into pB at
BamHI and PstI sites. The DNA sequences of these
recombinant cDNAs were confirmed by the dideoxynucleotide chain
termination method (24).
In Vitro Transcription and Translation--
Methods essentially
identical to those described previously were employed (23). T3
polymerase was used to obtain transcripts of these cDNAs subcloned
into pB vector. These were purified by phenol/chloroform extraction and
ethanol precipitation prior to use. Subsequently, the translation
reaction was carried out using the rabbit reticulocyte lysate (Promega)
in the presence of [3H]leucine,
[35S]methionine, [3H]myristic acid, or
[3H]acetyl-CoA under conditions recommended by the
manufacturer. The mixture (composed of 20.0 µl of rabbit reticulocyte
lysate; 1.0 µl of 1 mM leucine- or methionine-free amino
acid mixture or 1 mM complete amino acid mixture; 4.0 µl
of [3H]leucine (5 µCi), [35S]methionine
(1 µCi), [3H]myristic acid (25 µCi), or
[3H]acetyl-CoA (2 µCi); and 4.0 µl of mRNA) was
incubated at 30 °C for 90 min.
Transfection of COS-1 Cells and Determination of N-Myristoylated
Proteins--
The simian virus 40-transformed African Green monkey
kidney cell line, COS-1, was maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum (Life Technologies, Inc.). Cells (2 × 105)
were plated onto 35-mm diameter dishes 1 day before transfection. pcDNA3 construct (2 µg; Invitrogen, San Diego, CA) containing mutant TNF cDNA was used to transfect each plate of COS-1 cells along with 4 µl of LipofectAMINE (2 mg/ml; Life Technologies, Inc.)
in 1 ml of serum-free medium. After incubation for 5 h at 37 °C, the cells were refed with serum-containing medium and
incubated again at 37 °C for 24 h. The cells were then washed
twice with 1 ml of serum-free Dulbecco's modified Eagle's medium and
incubated for 5 h in 1 ml of Dulbecco's modified Eagle's medium
with 2% fetal calf serum containing [3H]myristic acid
(100 µCi/ml). Subsequently, the cells were washed three times with
Dulbecco's phosphate-buffered saline and collected with cell scrapers,
followed by lysis with 200 µl of radioimmune precipitation buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, proteinase inhibitors) on
ice for 20 min. The cell lysates were centrifuged at 15,000 rpm at
4 °C for 15 min in a microcentrifuge (Hitachi; model CF15D2), and
supernatants were collected. After immunoprecipitation with anti-TNF
antibody, the samples were analyzed by SDS-PAGE and fluorography.
Western Blotting--
TNF samples immunoprecipitated from
in vitro translation products or total cell lysates of each
group of transfected cells were resolved by 12.5% SDS-PAGE and then
transferred to an Immobilon-P transfer membrane (Millipore, Corp.).
After blocking with nonfat milk, the membrane was probed with a
specific goat anti-human TNF antibody as described previously (25).
Immunoreactive proteins were specifically detected by incubation with
horseradish peroxidase-conjugated anti-goat IgG antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). The membrane was developed with
ECL Western blotting reagent (Amersham Pharmacia Biotech) and exposed
to an x-ray film (Eastman Kodak Co.). Quantitative analysis of
immunoreactive proteins on the membrane was carried out using the
storage phosphor imaging screen and GS-250 Molecular Imager
(Bio-Rad).
Immunoprecipitation--
Samples containing TNF mutants were
immunoprecipitated with a specific goat anti-human TNF polyclonal
antibody (R & D systems) as described (23).
SDS-PAGE and Fluorography--
Samples were denatured by boiling
for 3 min in SDS-sample buffer followed by analysis by SDS-PAGE on a
12.5% gel. Thereafter, the gel was fixed and soaked in
AmplifyTM (Amersham Pharmacia Biotech) for 30 min. The gel
was dried under vacuum and exposed to an x-ray film (Kodak) for an
appropriate period. Quantitative analysis of the labeled proteins was
carried out by scanning the fluorogram using an imaging densitometer
(Bio-Rad; model GS-700).
Analysis of Bound Fatty Acids--
Fatty acid-labeled TNF
mutants immunoprecipitated from in vitro translation
products were resolved by 12.5% SDS-PAGE and then transferred to an
Immobilon-P transfer membrane. The region of membrane containing the
labeled TNF mutant, identified by Western blotting with anti-human TNF
antibody, was excised and hydrolyzed in 6 N HCl at
110 °C for 16 h. The released fatty acids were extracted in
hexane and run on a thin-layer chromatography plate (RP18; Merck) with
acetonitrile/acetic acid (9:1) as the solvent system. Radioactivity on
the thin layer plate was made visible by spraying with
En3Hance (PerkinElmer Life Sciences).
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RESULTS |
Amino Acid at Position 3 in the N-Myristoylation Consensus Motif
Strongly Affects Protein N-Myristoylation and N-Acetylation--
To
examine the amino-terminal sequence requirements for cotranslational
protein N-myristoylation, and to reveal the difference in
the N-terminal sequence requirement for protein
N-myristoylation and N-acetylation, the
N-terminal 9 residues of the mature domain of TNF including the
initiating Met were changed to the N-myristoylation consensus motif, and the susceptibility to cotranslational protein N-myristoylation and N-acetylation was evaluated
by an in vitro translation system. Since
pro-TNF, a
mature domain of TNF in which the initiating Met was introduced at the
N terminus, has Met and Ser residues at positions 1 and 6, respectively, Val at position 2 was replaced with Gly to obtain
V2G-TNF, in which the N-terminal 9 residues were adapted to the
N-myristoylation consensus motif,
Met-Gly-X-X-X-Ser-X-X-X
(Fig. 1).

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Fig. 1.
Schematic representation of V2G-TNF
generation. The cDNA coding for pro-TNF, which contains the
mature domain of TNF, was first generated from pro-TNF cDNA by
deleting the nucleotide sequence encoding the propeptide region of
pro-TNF. Subsequently, V2G-TNF cDNA was generated from pro-TNF
cDNA by site-directed mutagenesis.
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As shown in Fig. 2 (lanes
1 and 2), translation of mRNAs coding for
pro-TNF and V2G-TNF in the presence of [3H]leucine
gave rise to two translation products; one is the major product with an
expected molecular mass (17 kDa), and the other is a fainter band with
an ~2-kDa larger molecular mass. However, no incorporation of
[3H]myristic acid and [3H]acetyl-CoA was
detected in these translation products as shown in Fig. 2
(lanes 5, 6, 9, and
10).

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Fig. 2.
In vitro translation of mRNAs
coding for pro-, V2G-, R3A-, and R3D-TNF.
The mRNAs encoding pro-, V2G-, R3A-, and R3D-TNF were translated
in vitro in the presence of [3H]leucine,
[3H]myristic acid, or [3H]acetyl-CoA using
rabbit reticulocyte lysate. Following immunoprecipitation with anti-TNF
antibody, the labeled translation products were analyzed by SDS-PAGE
and fluorography.
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From early experiments performed by Towler et al. (2) using
purified yeast NMT and the synthetic peptide substrates, it has
been reported that an amino acid residue at position 3 affects the
susceptibility to protein N-myristoylation. We therefore
prepared two additional constructs, R3A- and R3D-TNF, in which Arg at
position 3 in V2G-TNF was replaced with Ala and Asp, respectively.
[3H]Leucine labeling revealed an efficient expression of
R3A- and R3D-TNF as observed with V2G-TNF (Fig. 2, lanes
3 and 4). In R3A-TNF, significant incorporation
of [3H]myristic acid, but not
[3H]acetyl-CoA, was observed (lanes
7 and 11). Conversely, significant incorporation
of [3H]acetyl-CoA, but not [3H]myristic
acid, was observed with R3D-TNF (lanes 8 and
12).
To determine whether the incorporation of [3H]myristic
acid into R3A-TNF was comparable with that into proteins having a
natural N-myristoylation motif, incorporation of
[3H]leucine and [3H]myristic acid into
R3A-TNF was compared with those into Gag-TNF and Gi1
-TNF
(22) in which the N-terminal 10 residues of the Gag protein or
Gi1
were linked to the N terminus of the mature domain
of TNF. As shown in Fig. 3A
(lanes 1-3 and 7-9), incorporations of [3H]leucine and [3H]myristic acid into
these three TNF mutants were found to be comparable, indicating that
R3A-TNF is efficiently myristoylated, similar to proteins having a
natural N-myristoylation motif. Next, we compared the
incorporation of [3H]acetyl-CoA into R3D-TNF with that
into ovalbumin, a naturally acetylated protein (17). In this case,
since specific antibody against ovalbumin was not available for the
immunoprecipitation of the in vitro translated products,
OVA60-TNF, in which the N-terminal 60 residues of ovalbumin were linked
to the N terminus of TNF, was used. As expected, only a low level of
[3H]leucine- and [3H]acetyl-CoA
incorporation was observed with ovalbumin, as shown in Fig.
3A (lanes 6 and 18). In
contrast, efficient [3H]leucine- and
[3H]acetyl-CoA incorporation was detected with OVA60-TNF
(lanes 5 and 17). As shown in Fig.
3A (lanes 4, 5,
16, and 17), incorporation of
[3H]leucine and [3H]acetyl-CoA into R3D-TNF
was comparable with those of OVA60-TNF, indicating that R3D-TNF is as
efficiently acetylated as the naturally acetylated protein. Analysis of
the 3H-labeled fatty acid attached to the R3A-TNF by TLC
confirmed the presence of [3H]myristic acid (Fig.
3B, lane 3). In contrast,
3H-labeled fatty acid attached to the R3D-TNF was not
detected on the TLC plate (lane 4). However,
acetic acid liberated from the acetylated protein is volatile and will
be evaporated by the extraction and concentration procedure. Therefore,
this result is consistent with the fatty acid attached to the R3D-TNF
being acetic acid. Taken together, it is suggested that the amino acid residue at position 3 in the
Met-Gly-X-X-X-Ser-X-X-X
motif strongly affects the susceptibility of the protein to two
different cotranslational protein modifications,
N-myristoylation and N-acetylation.

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Fig. 3.
Characterization of protein
N-myristoylation and N-acetylation of
TNF mutants and ovalbumin. The mRNAs encoding Gag-TNF,
Gi1 -TNF, R3A-TNF, R3D-TNF, OVA60-TNF, and ovalbumin were
translated in vitro in the presence of
[3H]leucine, [3H]myristic acid, or
[3H]acetyl-CoA using rabbit reticulocyte lysate. The
labeled translation products were analyzed directly (I.P.
) or following immunoprecipitation with anti-TNF antibody
(I.P. +) by SDS-PAGE and fluorography (A).
[3H]Fatty acids attached to the R3A- and R3D-TNF were
analyzed by thin layer chromatography. A [3H]fatty
acid-labeled protein band of R3A- or R3D-TNF on the transfer membrane,
identified by Western blotting, was excised, and the fatty acids were
liberated by acid treatment and extracted with hexane. The extracts
from R3A- and R3D-TNF (B, lanes 3 and
4, respectively), together with control
[3H]palmitic acid (lane 1),
[3H]myristic acid (lane 2), and
[3H]acetyl-CoA (lane 5), were
separated by thin layer chromatography and detected by fluorography.
The migration positions of control [3H]myristic acid
(Myr) and [3H]palmitic acid (Pal)
are indicated. The arrows indicate the origin
(B).
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As shown in Fig. 2 (lanes 3, 4,
7, and 12), protein N-myristoylation
and N-acetylation were specifically observed in the lower [3H]leucine-labeled band, with no modification of the
upper band observed. To clarify the basis for this, differential
labeling of these two protein bands with [3H]leucine,
[35S]methionine, [3H]myristic acid, and
[3H]acetyl-CoA was performed using
pro-, R3A-, and
R3D-TNF mRNA. In these three TNF variants, the initiating Met is
the only Met residue in the entire molecule; in contrast, these TNFs
contain several residues of Leu in their amino acid sequences. As shown in Fig. 4, [35S]methionine
was specifically incorporated into the upper band, whereas
[3H]leucine was incorporated into both bands.
Incorporation of [3H]myristic acid into R3A-TNF and
[3H]acetyl-CoA into R3D-TNF was observed exclusively in
the lower band. Since protein N-myristoylation and protein
N-acetylation occurs on Gly-2 after removal of the
initiating Met and there is no Met residue in the mature domain of TNF,
these results clearly indicated that the upper band corresponds to the
protein species retaining the initiating Met residue and the lower band
to the one lacking this residue.

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Fig. 4.
Differential labeling of the two distinct
protein bands obtained by in vitro translation of
mRNAs coding for pro-, R3A-, and
R3D-TNF. The mRNAs encoding pro-, R3A-, and R3D-TNF were
translated in vitro in the presence of
[3H]leucine, [35S]methionine,
[3H]myristic acid, or [3H]acetyl-CoA using
rabbit reticulocyte lysate. Following immunoprecipitation with anti-TNF
antibody, the labeled translation products were analyzed by SDS-PAGE
and fluorography.
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Effect of the Amino Acid Residue at Position 3 in the
N-Myristoylation Consensus Motif on the Efficiency of the
Cotranslational N-myristoylation and N-Acetylation Reaction--
To
investigate the amino acid requirement at position 3 in the
Met-Gly-X-X-X-Ser-X-X-X
motif for protein N-myristoylation and
N-acetylation, 20 mutants, each with a different amino acid at position 3, were generated, and their susceptibility to the two
cotranslational modifications was evaluated by the same method as
above. The results of 20 amino acids are arranged according to their
radius of gyration. All of these mutants were efficiently expressed as
determined by the incorporation of [3H]leucine as shown
in the upper panels of Fig.
5A. The ratio of the amount of
the two [3H]leucine-labeled protein bands was almost the
same in these 20 mutants, indicating that there is no significant
difference in the efficiency of the removal of the initiating Met
residue in these mutants.

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Fig. 5.
Effect of the amino acid residue at position
3 in N-myristoylation consensus motif on the
efficiency of cotranslational N-myristoylation
reaction. The mRNAs encoding R3X-TNF were translated in
vitro in the presence of [3H]leucine or
[3H]myristic acid using rabbit reticulocyte lysate.
Following immunoprecipitation with anti-TNF antibody, the labeled
translation products were analyzed by SDS-PAGE and fluorography.
Results of the 20 amino acids were arranged according to their radius
of gyration. Three independent experiments showed similar labeling
patterns (A). The efficiency of protein
N-myristoylation ([3H]myristic acid
incorporation/[3H]leucine incorporation) of R3X-TNF was
compared by quantitative analysis of the fluorogram of
[3H]myristic acid- and [3H]leucine-labeled
proteins shown in the upper and lower
panels of A. Relative N-myristoylation
efficiency of each R3X-TNF was expressed as percentage of the
myristoylation efficiency of R3A-TNF. Results of the 20 amino acids
were arranged according to their radius of gyration. Data are expressed
as mean ± S.D. of three independent experiments
(B).
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The labeling with [3H]myristic acid revealed a strong
correlation between the radius of gyration of the amino acid at
position 3 and the efficiency of protein N-myristoylation as
shown in the lower panels of Fig. 5A.
The relationship between the relative N-myristoylation
efficiency and the radius of gyration of amino acid at position 3 is
summarized in Fig. 5B. The presence of Gly, Ala, Ser, Cys,
Thr, Val, Asn, Leu, Ile, Gln, and His residues, each having a radius of
gyration smaller than 1.80 Å, at position 3 led to efficient
[3H]myristic acid labeling. In contrast, the presence of
amino acids with a radius of gyration larger than 1.80 Å (Phe, Lys,
Tyr, Trp, and Arg) at this position completely inhibited the
[3H]myristic acid incorporation. The presence of the Met
residue, which has an intermediate radius of gyration (1.80 Å) led to
a diminished efficiency of N-myristoylation. In addition to
the restriction by the radius of gyration of the amino acid, it was also revealed that the presence of negatively charged residues (Asp and
Glu) and Pro residue at this position completely inhibited the
myristoylation reaction. Labeling of these TNF mutants with [3H]acetyl-CoA revealed that nonmyristoylated mutants
with Asp or Glu at position 3 and a weakly myristoylated mutant having
Met at this position were efficiently acetylated as shown in the
lower panels of Fig.
6A. In addition, a low level
of [3H]acetyl-CoA incorporation was observed with an
effectively myristoylated mutant having Gly at position 3. These
results indicate that the amino acid at position 3 in the
Met-Gly-X-X-X-Ser-X-X-X
motif affected differently the two cotranslational protein
modifications, N-myristoylation and
N-acetylation.

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Fig. 6.
Effect of the amino acid residue at
position 3 on the protein N-myristoylation and
N-acetylation of R3X-TNF and R3X,S6A-TNF. The
mRNAs encoding R3X-TNF and R3X,S6A-TNF were translated in
vitro in the presence of [3H]leucine,
[3H]myristic acid, or [3H]acetyl-CoA using
rabbit reticulocyte lysate. Following immunoprecipitation with anti-TNF
antibody, the labeled translation products were analyzed by SDS-PAGE
and fluorography. Results of the 20 amino acids were arranged according
to their radius of gyration. A and B show results
with R3X-TNF and R3X,S6A-TNF, respectively.
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Effect of the Lack of a Ser Residue at Position 6 in the
N-Myristoylation Consensus Motif on Protein N-Myristoylation and
N-Acetylation--
It is generally accepted that Ser or Thr is
preferred at position 6 for protein N-myristoylation.
However, Ser or Thr at this position is neither sufficient nor critical
for protein N-myristoylation. Therefore, we next examined
the effect of a lack of the Ser residue at position 6 in the
N-myristoylation consensus motif on cotranslational protein
N-myristoylation and N-acetylation. In this
experiment, R3X,S6A mutants in which the Ser residue at position
6 in each of the R3X mutants was changed to Ala, were generated, and
their susceptibility to N-myristoylation and
N-acetylation reaction was determined by metabolic labeling.
As shown in the middle panels of Fig.
6B, dramatic changes in the amino acid requirement at position 3 were observed for protein N-myristoylation. The
number of amino acid residues that can direct protein
N-myristoylation was strikingly reduced; only 2 amino acids,
Asn and Gln, could direct efficient modification. In contrast, the
number of amino acid residues that direct protein
N-acetylation was increased to include Ser and Thr. These
results show that the amino acid residue at position 6 strongly affects
the amino acid requirement at position 3 for both protein
N-myristoylation and protein N-acetylation. The
susceptibility of R3X-TNF and R3X,S6A-TNF to protein
N-myristoylation and N-acetylation is summarized
in Fig. 7.

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Fig. 7.
Summary of cotranslational
N-myristoylation and N-acetylation of
R3X-TNF and R3X,S6A-TNF. Susceptibility of R3X-TNF and R3X,S6A-TNF
to protein N-myristoylation and N-acetylation as
determined by the metabolic labeling experiments in Fig. 6 is
summarized. ++, effectively modified; +, weakly modified; ±, slightly
modified.
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Amino Acid Requirements for Protein N-Myristoylation Found in the
in Vitro Translation System Are Applicable to the Protein
N-Myristoylation in Intact Cells--
To determine whether the amino
acid requirements for protein N-myristoylation found in the
in vitro translation system are applicable to the
N-myristoylation reaction in intact cells, R3X mutants were
expressed in COS-1 cells, and their susceptibility to
N-myristoylation was evaluated by metabolic labeling with
[3H]myristic acid. The same experiment with
[3H]acetyl-CoA could not be performed because of the
limited supply of [3H]acetyl-CoA. As shown in the
upper panels of Fig.
8, Western blotting analysis of the
expressed proteins immunoprecipitated from the total cell lysates
revealed that a remarkable difference in the level of protein
expression was present within these mutants. Two mutants having a Cys
or Val residue at position 3 were not expressed in COS-1 cells.
Interestingly, only low levels of protein expression were equally
observed with the mutants in which the radius of gyration of amino
acids at position 3 is larger than 1.80 Å, namely Phe, Lys, Tyr, Trp,
and Arg. In contrast to the results obtained by the in vitro
translation system, only a single protein band with an expected
molecular mass was detected in all these mutants in the Western
blotting analysis, indicating that the initiating Met residue in these
mutants was completely removed in intact cells.

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Fig. 8.
N-myristoylation of R3X-TNF
expressed in COS-1 cells. The cDNAs encoding R3X-TNF were
transfected into COS-1 cells, and their expression and
N-myristoylation were evaluated by Western blotting analysis
and [3H]myristic acid labeling, respectively. Results of
the 20 amino acids were arranged according to their radius of
gyration.
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Incorporation of [3H]myristic acid into these COS-1 cells
is shown in the lower panels of Fig. 8. As for
the mutants in which the expression of the protein was observed,
exactly the same patterns of [3H]myristic acid
incorporation as with the in vitro translation system were
observed. These results indicated that the amino acid requirements at
position 3 in the N-myristoylation consensus motif found in
the in vitro translation system are consistent with those observed for the N-myristoylation reaction in intact cells.
A Lysine Residue Is Permitted at Position 3 in the Naturally
Occurring N-Myristoylation Motif--
To determine whether the amino
acid requirements at position 3 for protein N-myristoylation
found in the present study are consistent with the amino acids at this
position in naturally occurring N-myristoylation motifs, the
number of each amino acid residue located at position 3 in 88 N-myristoylated proteins listed in two recent reviews (5, 6)
were counted and summarized in Fig. 9.
Ninety percent (79 of 88) of these proteins had amino acid residues at
position 3 that were consistent with the amino acid requirements at
position 3 for protein N-myristoylation found in this study.
Seven out of nine proteins in which the amino acid at position 3 is
inconsistent with our present results have a Lys residue at this
position. Thus, Lys residue seems to be permitted at position 3 in the
naturally occurring myristoylation motif.

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Fig. 9.
Amino acid residues at position 3 in
naturally occurring N-myristoylation motifs. The
number of each amino acid residue located at position 3 in 88 N-myristoylated proteins listed in two recent reviews by
Boutin (5) and Resh (6) were counted and arranged according to their
radius of gyration. , amino acid residue consistent with the amino
acid requirements at position 3 found in this study. , amino acid
residue inconsistent with the amino acid requirements at position 3 found in this study.
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One possible explanation for this discrepancy might be our use of a
nonmyristoylated model protein to study the amino acid requirement
instead of using a naturally myristoylated protein. To test this
possibility, the amino acid at position 3 in Gag-TNF and
Gi1
-TNF, which have a natural myristoylation motif at
the N terminus, was changed to Lys, and the susceptibility to
N-myristoylation was evaluated. As shown in Fig.
10 (lanes 1-4),
myristoylation of Gag-TNF and Gi1
-TNF was remarkably
reduced by replacing the amino acid at position 3 with Lys; only a very
low level of [3H]myristic acid incorporation was observed
with Gag-Q3K-TNF and Gi1
-C3K-TNF. These results clearly
indicated that the discrepancy in the amino acid requirement at
position 3 was not due to the use of a nonmyristoylated model protein.
Interestingly, however, when the N-terminal 10 residues of the mature
domain of TNF were changed to those of Arf6 or hippocalcin, both having
Lys-3 in their myristoylation motif, remarkable myristoylation was
detected as with Gag-TNF or Gi1
-TNF as shown in Fig. 10
(lanes 7 and 8). These results
suggested that the N-myristoylation motif having Lys-3, such
as that of Arf6 or hippocalcin, might have some specific structural
determinant that permits the Lys residue at position 3, while still
directing protein N-myristoylation.

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Fig. 10.
Effect of a Lys residue at position 3 in the
N-myristoylation consensus motif on protein
N-myristoylation. The mRNAs encoding Gag-,
Gag-Q3K-, Gi1 -, Gi1 -C3K-, R3A-, R3K-,
Arf6-, and hippocalcin-TNF were translated in vitro in the
presence of [3H]leucine or [3H]myristic
acid using rabbit reticulocyte lysate. Following immunoprecipitation
with anti-TNF antibody, the labeled translation products were analyzed
by SDS-PAGE and fluorography.
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DISCUSSION |
Since all protein synthesis begins at the N terminus, this region
provides an initial and important site of cotranslational protein
processing. Removal of the initiator methionine and modification of the
-amino group are examples of commonly encountered N-terminal modifications. Three of these modifications, excision of the initiator methionine, N-acetylation, and N-myristoylation,
potentially affect many of the eukaryotic cytoplasmic proteins and
variously correlate with their stability, physiological function, and
degradation. These three modifications are catalyzed by methionine
aminopeptidases, N-acetyltransferases, and NMTs, respectively.
Methionine aminopeptidases involved in the cotranslational removal of
the initiator Met are defined by a highly conserved substrate
specificity, which is dictated by the residue adjacent to the Met
residue (26, 27). The 7 amino acids that have the smallest radii of
gyration are substrates for methionine aminopeptidases, while those
with the 13 largest side chains are not. Residues downstream of the
specificity-determining residue have little impact on the reaction.
N-Acetyltransferases that catalyze cotranslational protein
N-acetylation also have a restricted number of substrates
(28-31). Studies both in yeast and in higher eukaryotic cells showed
that the principal substrates of N-acetyltransferases are
proteins that have N-terminal Gly, Ala, Ser, or Thr residues (GAST
substrates), or Met residue (M substrates) (32-34). Unlike the
methionine aminopeptidases, this specificity is clearly affected by
downstream residues, making the rules that guide acetylation less
clear. In the case of M substrates, N-acetylation only
occurs if the adjacent residue is an Asp, Glu, or Asn. However, the
amino acid requirements for the adjacent residue of the GAST substrates
have not been defined as yet.
Protein N-myristoylation is a cotranslational protein
modification catalyzed by two enzymes, methionine aminopeptidase and NMT. Proteins destined to become myristoylated begin with the sequence
Met-Gly. The initiating methionine is removed cotranslationally by
methionine aminopeptidase, and then myristic acid is linked to Gly-2
via an amide bond by NMT. NMT catalyzes the transfer of myristic acid
from myristoyl-CoA to the N terminus of protein substrates. However,
not all proteins with an N-terminal glycine are
N-myristoylated, and the ability to be recognized by NMT
depends on the downstream amino acid sequence.
Some amino acid preferences have been observed at distinct positions
downstream of the N-terminal glycine (2, 13, 15). In general, Ser or
Thr is preferred at position 6, and N-terminal consensus motifs such as
Met-Gly-X-X-X-Ser/Thr-X-X
(13) or
Met-Gly-X-X-X-Ser/Thr-X-X-X (5) have been defined. In addition to the preference for a Ser/Thr
residue at position 6, positively charged residues (Lys or Arg) are
known to be preferred at position 7 and/or 8 (2, 15). These amino acid
preferences were confirmed by recent studies on the NMT structure as
determined by x-ray crystallography (35). In this study, the structure
of the yeast Saccharomyces cerevisiae NMT1p was solved as a
ternary complex and revealed how myristoyl-CoA and peptide substrates
bind to the enzymes. The determined structure allows identification of
specific residues within NMT that serve to restrict substrate
specificity to 14-carbon fatty acids and account for the preference of
Gly-2, Ser-6, and basic amino acids at positions 7 and 8 of the peptide substrate.
In the present study, to examine the amino-terminal sequence
requirements for the cotranslational N-myristoylation of
proteins, several series of mutants were prepared by site-directed
mutagenesis of the N-terminal region of the protein, using TNF as a
nonmyristoylated model protein. Subsequently, the susceptibility of
these mutants to the cotranslational N-myristoylation
reaction was evaluated by an in vitro
transcription/translation system using the rabbit reticulocyte lysate.
It was found that the radius of gyration of the amino acid residue at
position 3 in an N-myristoylation consensus motif,
Met-Gly-X-X-X-Ser-X-X-X,
strongly affected the cotranslational protein
N-myristoylation. Amino acids with a radius of gyration
larger than 1.80 Å (Phe, Lys, Tyr, Trp, and Arg) could not direct
efficient protein N-myristoylation in the in
vitro translation system. From the study on the x-ray
crystallography of NMT, it was proposed that during the catalytic
process, the amine nucleophile of the N-terminal Gly of the protein
substrate must move in the active site of NMT toward the thioester
carbonyl of myristoyl-CoA, which is positioned in the oxyanion hole
formed by the main chain amide NH groups of Phe-170 and Leu-171 (35). Therefore, the presence of amino acids having large radii of gyration at position 2 might cause a steric hindrance of the movement of the
N-terminal Gly. This notion is supported by the finding that the
presence of Pro at this position completely inhibits the reaction.
As mentioned earlier, similar limitation by the radius of gyration of
the amino acid residue penultimate to the N-terminal residue was
observed on the substrate specificity of methionine aminopeptidases.
Since both NMTs and methionine aminopeptidases function
cotranslationally while the nascent polypeptide chain is still attached
to the ribosomes (20, 36, 37), it might be possible to speculate that
these two enzymes may share similar restrictions with respect to their
substrate specificities.
In addition to the restriction by the radius of gyration of the amino
acid, the presence of either negatively charged residues (Asp, Glu) or
Pro at this position completely inhibited protein N-myristoylation. Labeling of these TNF mutants with
[3H]acetyl-CoA revealed that nonmyristoylated mutants
having an Asp or Glu residue at position 3 and weakly myristoylated
mutant having Met at this position were efficiently acetylated. These results clearly indicated that the amino acid at position 3 in the
Met-Gly-X-X-X-Ser-X-X-X
motif strongly affected two different cotranslational protein
modifications, N-myristoylation and
N-acetylation. Most of the cotranslationally modified TNF
mutants were modified exclusively with one of the two modifications,
indicating that the amino acid requirements for protein
N-myristoylation and protein N-acetylation are
generally different. However, some partially N-myristoylated
mutants such as R3G-TNF and R3M-TNF were found to be partially
N-acetylated, suggesting that the N-terminal sequence requirements for the two cotranslational modifications partially overlap.
When Ser at position 6 in the
Met-Gly-X-X-X-Ser-X-X-X
motif was replaced with Ala, amino acid requirements at position 3 for the two cotranslational modifications were dramatically changed. As for
protein N-myristoylation, only two amino acids, Asn and Gln,
could direct this modification. This result clearly indicates that the
amino acid requirements at position 3 for protein
N-myristoylation are significantly affected by the
downstream residue(s).
In contrast, in the case of protein N-acetylation, two
additional amino acids, Ser and Thr, could direct efficient protein modification. Two different interpretations can be derived to explain
this phenomenon. It may simply imply that the amino acid requirement at
position 3 for protein N-acetylation is affected by the
amino acid residue at position 6, as is the case with protein N-myristoylation. Alternatively, the
N-myristoylation reaction predominates over the
N-acetylation reaction, and inhibition of the
N-myristoylation reaction by replacing Ser-6 with Ala
resulted in the two N-terminal sequences (Gly-Ser- and Gly-Thr-) being N-acetylated. Further analysis is required to fully
characterize these experimental observations.
It is also interesting to investigate whether the amino acid
requirements at position 3 observed for R3X-TNF and R3X,S6A-TNF for protein N-acetylation are applicable to the other GAST
substrates of N-acetyl transferase. We are currently
investigating these issues.
Another issue to be clarified is whether the amino acid requirements
for protein N-myristoylation and N-acetylation
found in the in vitro translation system are fully
applicable in intact cells. As shown in Fig. 8, metabolic labeling of
COS-1 cells transfected with R3X-TNF revealed that the amino acid
requirements at position 3 in the N-myristoylation consensus
motif found in the in vitro translation system are
consistent with those observed in the N-myristoylation reaction in intact cells. Therefore, it is quite possible that the
amino acid requirements for both protein N-myristoylation and N-acetylation found in the in vitro
translation system are applicable to intact cells. In fact, when
N-terminal sequences of 88 N-myristoylated proteins with the
Met-Gly-X-X-X-Ser/Thr-X-X-X motif listed in two recent reviews (5, 6) were examined, 90% (79 of
88) of the amino acid residues at position 3 of these proteins were
found to be consistent with the amino acid requirements at position 3 for protein N-myristoylation found in this study. Analysis
of the N-myristoylation motifs in which the amino acid at
position 3 is inconsistent with our present results revealed that Lys
residue could be permitted at position 3 in the naturally occurring
N-myristoylation motif. In this case, however, it seems likely that N-myristoylation motifs having Lys-3 have some
specific structural determinant that permits the Lys residue at
position 3, while still directing protein N-myristoylation.
We are currently searching for the specific determinant in these
N-myristoylation motifs. In addition, when
N-myristoylated proteins with N-terminal Met-Gly-X-X-X-Ala-X-X-X-
sequences listed in these reviews were examined, four out of five
proteins were found to possess Asn or Gln residue at position 3. These
results strongly suggest that the amino acid requirements found in this
study correlated well with those for the myristoylation reaction in
intact cells.
Thus, analysis of amino acid requirements for cotranslational protein
modifications in the in vitro translation system is an
effective strategy for characterization of the consensus amino acid
sequences that direct cotranslational protein modifications.