Plant Physiol. (1998) 117: 949-960
Phosphoglycerylethanolamine Posttranslational Modification of
Plant Eukaryotic Elongation Factor 1
1
Wendy D. Ransom,
Pao-Chi Lao,
Douglas A. Gage, and
Wendy F. Boss*
Botany Department, North Carolina State University, Raleigh, North
Carolina 27695-7612 (W.D.R., W.F.B.); Department of Environmental and
Occupational Health, National Cheng Kung Medical College, Tainan 70428, Taiwan, Republic of China (P.-C.L.); and Department of Biochemistry,
Michigan State University, East Lansing, Michigan 48824 (D.A.G.)
 |
ABSTRACT |
Eukaryotic elongation factor 1
(eEF-1A) is a multifunctional protein. There are three known
posttranslational modifications of eEF-1A that could potentially affect
its function. Except for phosphorylation, the other posttranslational
modifications have not been demonstrated in plants. Using
matrix-assisted laser desorption/ionization-mass spectrometry and
peptide mass mapping, we show that carrot (Daucus carota
L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the
predominant ethanolamine-labeled protein in Arabidopsis seedlings and
tobacco (Nicotiana tabacum L.) cell cultures. In
vivo-labeling studies using [3H]glycerol,
[32P]Pi, [14C]myristic acid, and
[14C]linoleic acid indicated that the entire phospholipid
phosphatidylethanolamine is covalently attached to the protein. The PGE
lipid modification did not affect the partitioning of eEF-1A in Triton
X-114 or its actin-binding activity in in vitro assays. Our in vitro
data indicate that this newly characterized posttranslational
modification alone does not affect the function of eEF-1A. Therefore,
the PGE lipid modification may work in combination with other
posttranslational modifications to affect the distribution and the
function of eEF-1A within the cell.
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INTRODUCTION |
eEF-1A is an abundant protein that constitutes 1 to 3% of the
total soluble protein in the cell (Merrick and Hershey, 1996
). Several
different activities have been reported for this protein, ranging from
its well-established role in protein synthesis (Merrick and Hershey,
1996) and its ability to bind and bundle actin (Demma et al., 1990;
Edmonds et al., 1995), activate phosphatidylinositol 4-kinase (Yang et
al., 1993), and bind (Durso and Cyr, 1994; Durso et al., 1996) and
sever microtubules (Shiina et al., 1994), to its involvement in
ubiquitin-dependent degradation of N-acetylated protein
(Gonen et al., 1994).
Consistent with a predicted role of eEF-1A in regulating cytoskeletal
structure, in situ studies have shown that eEF-1A is associated with
both F-actin (Dharmawardhane et al., 1991; Collings et al., 1994; Clore
et al., 1996; Sun et al., 1997) and tubulin (Durso and Cyr, 1994; Durso
et al., 1996). Importantly, the distribution of eEF-1A changes when
Dictyostelium discoideum. is stimulated with cAMP
(Dharmawardhane et al., 1991). After adding cAMP,
there is a rapid (within 25 s) decrease in eEF-1A associated with
F-actin. As new filopodia form (by 90 s), eEF-1A relocalizes to
the filopodia F-actin. Recent work (Edmonds et al., 1995; Liu et al.,
1996; Murray et al., 1996) has shown that slight changes in pH will affect the actin binding, bundling, and translational activity of
eEF-1A. This work suggests that eEF-1A is a very sensitive monitor of
the status of protein synthesis and the cytoskeletal network. If eEF-1A
serves as an internal cell sensor, changing its location within the
cell, then there must be a mechanism regulating eEF-1A. One mechanism
by which both the function and distribution of a protein can be altered
is posttranslational modification.
At least three types of posttranslational modifications have been
reported so far for eEF-1A: Lys residues are methylated (Hiatt et al.,
1982; Van Hemert et al., 1984; Fonzi et al., 1985; Amons et al., 1993),
Ser residues are phosphorylated (Yang et al., 1993), and Glu residues
are modified by the attachment of PGE (Tisdale and Tartakoff, 1988;
Dever et al., 1989; Rosenberry et al., 1989; Whiteheart et al., 1989).
Even though the amino acid sequence of all eEF-1As remains highly
conserved, the presence and/or location of the different
posttranslational modifications are not (Cavallius et al., 1993;
Browning, 1996). The number and type of methylation (mono, dimethyl, or
trimethyl) vary depending on species (Dever et al., 1989). The PGE
modification reported in rabbit, mouse, and human was not found in
yeast (Cavallius et al., 1993). Although phosphorylation of the
elongation factor complex is associated with increased translational
activity (Venema et al., 1991; Chang and Traugh, 1997), to our
knowledge, only one function of eEF-1A has been correlated with the
requirement for a posttranslational modification. eEF-1A must be
phosphorylated to be a functional activator of PI-4 kinase (Yang et
al., 1993; Yang and Boss, 1994).
We have investigated the PGE posttranslational modification of eEF-1A.
The PGE modification has the potential for facilitating association of
eEF-1A with lipids or membranes. To study the PGE modification we
labeled carrot (Daucus carota L.) suspension-cultured cells
with [14C]ethanolamine. The incorporation of
ethanolamine into proteins is a rare event and has only been observed
in two types of proteins: those with GPI anchors (Aitken, 1992) and
eEF-1A with a PGE attachment (Tisdale and Tartakoff, 1988; Dever et
al., 1989; Rosenberry et al., 1989; Whiteheart et al., 1989). We will
show the incorporation of [14C]ethanolamine
into eEF-1A as part of a PGE modification and provide evidence for a
phospholipid posttranslational modification, which could provide a
mechanism for regulating the distribution of this multifunctional
protein within the cell.
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MATERIALS AND METHODS |
Wild carrot (Daucus carota L.) cells were grown in
suspension culture and transferred weekly as previously described
(Chen and Boss, 1990). Chicken muscle actin, TCA, and Triton X-114 were purchased from Sigma. QMA Accell Plus and C18 Accell Sep-Pak cartridges were purchased from Waters. [14C]Ethanolamine
(55 mCi/mmol), [14C]myristic acid (55 mCi/mmol), [3H]glycerol (20 Ci/mmol), and
[32P]Pi (40 mCi mL
1)
were purchased from American Radiolabeled Chemicals, Inc. (St. Louis,
MO). [14C]Linoleic acid (55.6 mCi/mmol) and
En3hance were purchased from DuPont NEN.
[3H]Palmitoleic acid (60 Ci/mmol) was a gift
from Dr. Leo Parks (North Carolina State University, Raleigh).
Arabidopsis (ecotype Landsberg) seeds were grown in Murashige and
Skoog liquid medium for 12 d, rotating at 1 rpm, at 23 ± 2°C under constant light from cool-white fluorescent 40-W bulbs.
L-(Tosylamido-2-phenyl)ethyl chloromethyl ketone-treated
trypsin was purchased from Worthington Biochemical Corporation
(Freehold, NJ). Butylated hydroxytoluene was purchased from Calbiochem.
TFA sequanal grade and Micro BCA protein assay reagent were purchased
from Pierce. Bradford protein assay reagent was purchased from Bio-Rad.
A phosphor imager (445SI, Molecular Dynamics) and a phosphor imager
tritium screen were used for some of the radioisotopic analysis.
Protein Purification
A rapid method was developed for purifying eEF-1A protein from a
small amount of cells using a modification of the method used by Yang
et al. (1993). The cells were harvested by filtration (gravity) on
Whatman No. 1 paper, weighed, and homogenized using ice-cold buffer at
1 mL/g fresh weight (buffer A: 30 mM Tris, 2 mM
EGTA, 1 mM EDTA, 1 mM sodium molybdate, 25%
glycerol [v/v], 1 mM PMSF, and 10 mM
-mercaptoethanol). All subsequent procedures were performed at 4°C
unless noted otherwise. The homogenate was centrifuged at
700g for 5 min. The 700g supernatant was
centrifuged at 40,000g, and the resulting supernatant was
used as the soluble-protein fraction. eEF-1A was purified from the
40,000g supernatant using a Sep-Pak Accell QMA Classic
cartridge (short body) attached to a 3-cc syringe. The cartridge was
equilibrated with 3 mL of buffer B (30 mM Tris-HCl, pH 7.2)
by adding buffer to the syringe and forcing it through the column by
hand. The 40,000g (7.3 mg/mL, 500 µL) supernatant was
placed in the syringe and forced through the cartridge. The protein in
the 40,000g supernatant was determined using the modified
Bradford assay (Bio-Rad) with BSA as a standard. After applying the
protein, two additional 500-µL aliquots of buffer B were forced
through the syringe and collected in 1.5-mL Eppendorf tubes to elute
eEF-1A (fractions 2 and 3). Remaining protein was eluted with 500 µL
of 100 mM KCl (buffer C). To remove noncovalently bound
lipids, protein samples were extracted in acidic chloroform:methanol as
previously described by Chen and Boss (1990). For trypsin digestion and
MALDI-MS analysis the purification was scaled up to use 12-cc Sep-Pak
Vac Accell QMA cartridges by increasing 2-fold the amount of
40,000g supernatant applied to the cartridge and by using a
vacuum to facilitate elution. The 12-cc Sep-Pak Vac Accell QMA
cartridge was equilibrated with buffer B, the 40,000g
supernatant was applied, and the column was eluted with 6 mL of buffers
B and C.
Isolation of cytoskeleton-associated eEF-1A was performed as described
above, except for the use of a different homogenization buffer modified
from Abe et al. (1992). The buffer contained 5 mM Hepes, pH
7.0, 10 mM MgCl2, 2 mM
EGTA, 1 mM PMSF, and 1 mg/100 mL leupeptin (buffer D).
Isolation of Plasma Membranes
Cells were homogenized in buffer A as described above and
centrifuged at 700g for 5 min. The 700g
supernatant was centrifuged at 40,000g for 1 h and the
resulting pellet was used as the microsomal fraction. Plasma membranes
were further purified from the 40,000g pellet by aqueous
two-phase partitioning (Wheeler and Boss, 1987). Protein concentration
was determined using the microbicinchoninic acid assay.
In Vivo Labeling
Two days after transfer, carrot cells were incubated with
[14C]ethanolamine (5-13 µCi/1 g fresh
weight), [14C]myristic acid (3.6 µCi/g fresh
weight), [3H]glycerol (3.5 µCi/g fresh
weight), [14C]linoleic acid (8.8 mCi/g fresh
weight), or [3H]palmitoleic acid (35 µCi/g
fresh weight) for 48 h. The carrot cells were harvested and
homogenized using buffer A, and soluble protein, purified eEF-1A,
microsomes, and plasma membranes were obtained as described above.
Arabidopsis (ecotype Landsberg) seedlings were grown in
Murashige and Skoog liquid culture for 12 d. Plants were incubated with [14C]ethanolamine (3.5 µCi/3-4 plants)
in the liquid medium for 2 d. Plants were rinsed with fresh medium
and harvested. The roots were excised from the shoots and leaves. The
material was homogenized using buffer A, centrifuged at 700g
for 6 min, and the supernatant was centrifuged at 40,000g
for 1 h. Aliquots of soluble and microsomal proteins were analyzed
by SDS-PAGE, or precipitated with TCA and delipidated as described
below. For radioisotopic analysis of SDS-PAGE the polyacrylamide gel
was treated with En3hance, dried, and exposed to
film.
Tobacco (Nicotiana tabacum cv Wisconsin 38)
suspension-cultured cells were grown as described in Roberts et al.
(1992). Two days after transfer, cells were incubated with
[14C]ethanolamine (4 µCi/g fresh weight) for
two d. The cells were harvested and homogenized using buffer A, as
described for carrot cells. A 40,000g supernatant and
microsomal fraction was obtained for western-blot analysis using an
antibody to D. discoideum eEF-1A (1:500 dilution of the
antiserum; Demma et al., 1990). The western blot was exposed to film
for 1 month.
Analysis of eEF-1A Peptides
[14C]et-eEF-1A
([14C]et-eEF-1A) and nonradioactive eEF-1A from
carrot cells were excised from 10% SDS-polyacrylamide gels and digested with L-(tosylamido-2-phenyl)ethyl chloromethyl
ketone-treated Trypsin (1:25, w/w) at 30°C for 24 h as described
by Stone and Williams (1993). The resulting eEF-1A peptides were
concentrated in a SpeedVac to 200 µL. The concentrated peptides were
loaded onto a C18 Sep-Pak Classic cartridge of the short-body type (500 µg/200 µL) that had been pre-equilibrated with methanol (7 mL) and
then with deionized water (7 mL). The peptides were eluted with 80%
acetonitrile in 0.1% TFA. The eluted peptides were concentrated to 200 µL in a SpeedVac and separated on an Ultramex 5 C18 HPLC column
(250 × 10 mm, Phenomenex, Torrance, CA) using a nonlinear gradient of 0 to 80% acetonitrile in 0.1% TFA at a flow rate of 0.8 mL/min for 200 min. From time 0 to 5 min a 0% gradient was used, and
from 5 to 200 min a 0 to 80% gradient was used. Elution of the
[14C]et-eEF-1A peptides was determined by
counting an aliquot (200 µL) of each 1-mL fraction collected. Each
aliquot was placed in a scintillation vial with Scintiverse II cocktail
and counted in a scintillation counter (model LS 7000, Beckman). The
fractions containing [14C]ethanolamine were
analyzed by MALDI-MS.
MS
MALDI-MS analyses were performed on a Voyager Elite time-of-flight
instrument (PerSeptive Biosystems, Framingham, MA) equipped with a
N2 laser (337 nm, 2-ns pulse width). Data were
acquired in the linear continuous-extraction mode with an acceleration potential of 25 kV. Samples were prepared for analysis by mixing 1 µL
of the concentrated HPLC fraction with 1 µL of a saturated solution
of 4-hydroxy--cyanocinnamic acid matrix in 1:1 acetonitrile: 0.1%
aqueous TFA in a well on the sample stage. The solution was allowed to
air dry before the sample stage was introduced through a vacuum lock
into the instrument. Spectra were produced by averaging transients from
64 laser shots.
TCA Precipitation of [14C]et-eEF-1A
A method was developed for rapid analysis of covalently bound
[14C]et-eEF-1A. We precipitated the protein
with TCA and then removed noncovalently bound lipid using acetone.
Samples (5-20 µg of protein) were spotted onto 1-cm square pieces of
Micro Filtration Systems 1514A paper (Pleasanton, CA), placed in a
beaker containing ice-cold TCA (5%, w/v; 200 mL/20 filters), and
incubated for 5 min on a flat rotary shaker at 100 rpm and 25°C. The
TCA was discarded and the filters were washed two more times with an
equal volume of ice-cold TCA. After removing the final TCA wash,
ice-cold acetone (200 mL/20 filter papers) was added to the beaker to
remove noncovalently bound lipid, and filters were washed for 5 min at
200 rpm and 25°C. The acetone was discarded and the filters were
washed again with an equal volume of ice-cold acetone. Each filter was
dipped sequentially into a second beaker containing ice-cold acetone and then ether. The washed filters were air dried, placed in a scintillation vial with Scintiverse II cocktail (Fisher Scientific), and counted in a Beckman LS 7000 scintillation counter. Data are reported as dpm recovered minus a control of the same size filter paper
that was incubated in the same beaker throughout the entire procedure
to determine nonspecific binding to the filter paper. Additional
controls were done to determine whether each of the isotopes incubated
with protein would nonspecifically precipitate and be recovered in the
delipidated product.
To test for removal of noncovalently bound lipid, we added
[14C]PC to nonradiolabeled protein prior to TCA
precipitation. By the third acetone wash, no
[14C]PC was detected in the wash and only 23 dpm of the original 13,100 dpm added were recovered with the final
washed precipitate (Table I). The entire
acetone wash was dried, Scintiverse II cocktail was added, and the wash
was counted in a Beckman LS 7000 scintillation counter. Other controls
were [14C]ethanolamine plus nonradiolabeled
eEF-1A, [14C]myristic acid plus nonradiolabeled
eEF-1A, [3H]glycerol plus nonradiolabeled
eEF-1A, and [3H]inositol plus
nonradiolabeled eEF-1A (Table I). These controls were spotted onto
filter paper and washed as described above. The same result observed
for [14C]PC was obtained with the other
controls except for [3H]inositol. When
[3H]inositol was added to nonradiolabeled
eEF-1A, the residual dpms indicated that noncovalently bound inositol
was recovered. Therefore, this technique was not used for evaluating
[3H]inositol incorporation into eEF-1A.
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Table I.
A rapid method for analyzing isotope incorporation
into eEF-1A
[14C]PC, [14C]ethanolamine,
[14C]myristate, and [3H]glycerol do not
bind nonspecifically to eEF-1A or to catalase. [14C]PC
was mixed with catalase (50 µg of protein), precipitated with TCA,
and delipidated as described in ``Materials and Methods''.
Nonradioactive eEF-1A (5 µg of protein) was mixed with
[14C]ethanolamine, [14C]myristate, or
[3H]glycerol, TCA precipitated, and delipidated as
described in ``Materials and Methods''. [14C]et-eEF-1A
(2 µg of protein) was TCA precipitated and delipidated as described
in ``Materials and Methods''. The numbers are the average of four
samples for [14C]PC, two samples for
[14C]ethanolamine, and three samples for
[14C]myristic acid, [3H]glycerol,
[3H]inositol, and [14C]et-eEF-1A.
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Actin-Binding Assay
The actin-binding assay was performed according to the procedure
of Demma et al. (1990) with minor modifications. A mixture of chicken
muscle actin (0.2 or 0.4 mg/mL) and
[14C]et-eEF-1A (0.1 mg/mL) or BSA (0.2 mg/mL),
actin alone, or [14C]et-eEF-1A alone was
incubated at room temperature for 30 min in 100 µL of actin
sedimentation buffer (2 mM MgCl2, 2 mM EGTA, 50 mM KCl, 30 mM Tris-HCl,
pH 7.2, 1 µM CaCl2, and 1 mM ATP). The incubated samples were centrifuged in a
tabletop microfuge at 15,000 rpm for 30 min. The supernatant was
removed and the actin pellet was resuspended in 20 µL of
sedimentation buffer. The supernatant and pellet were analyzed by
SDS-PAGE and exposed to a phosphor imager tritium screen for 4 weeks.
Triton X-114 Partitioning
Triton X-114 partitioning was performed as described by Bordier
(1981). The Triton X-114 was precondensed before use with Tris buffer
and butylated hydroxytoluene (Bordier, 1981). The protein samples (15 µg) were prepared in 200 µL of TBS buffer (10 mM
Tris-HCl, pH 7.5, 150 mM NaCl) and 0.5% Triton X-114 (v/v) at 0°C. For separation of the proteins, the protein in TBS buffer plus Triton X-114 was layered onto a 6% Suc cushion consisting of 6%
Suc (w/v), 10 mM Tris-HCl, pH 7.4, 150 mM NaCl,
and 0.6% Triton X-114 in a 1.5-mL Eppendorf microfuge tube. The tube
was incubated at 30°C for 5 min then centrifuged for 5 min at
300g. The detergent-rich phase collects as an oily drop at
the bottom of the tube. The aqueous phase was removed to a clean
Eppendorf tube and mixed with fresh 0.5% Triton X-114. The aqueous
phase was incubated at 0°C for 5 min and then relayered onto the Suc cushion, incubated at 30°C for 5 min, and centrifuged for 5 min at
300g. The aqueous phase was removed to a clean Eppendorf
tube and mixed with fresh 2% Triton X-114, incubated at 30°C for 5 min, and centrifuged for 5 min at 300g. The aqueous phase
was removed to a clean Eppendorf tube, and the protein was precipitated with ice-cold acetone overnight at 
20°C and centrifuged at 15,000 rpm for 15 min. The Suc cushion was removed and discarded. The detergent-rich phase and the acetone-precipitated aqueous phase were
analyzed by SDS-PAGE and exposed to a phosphor imager tritium screen
for 11 d.
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RESULTS |
[14C]Ethanolamine Is Incorporated into a 49-kD
Peptide in Plants
To determine whether [14C]ethanolamine is
incorporated into plant proteins, Arabidopsis plants and carrot
suspension-cultured cells were incubated with
[14C]ethanolamine for 2 d. When proteins
from carrot cells were analyzed by SDS-PAGE and autoradiography, only
one polypeptide band had incorporated the
[14C]ethanolamine (Fig.
1). The apparent molecular mass of the
[14C]-labeled band was 49 kD based on molecular
mass markers (Fig. 1). The
[14C]ethanolamine-labeled protein copurified
with carrot eEF-1A, and was recognized by antibodies to D. discoideum eEF-1A (Fig. 1).
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| Figure 1.
Carrot cells were incubated with
[14C]ethanolamine (7 µCi/g fresh weight) and harvested
as described in ``Materials and Methods''. The 40,000g
carrot supernatant (lanes 1, 3, and 5) and purified eEF-1A (lanes 2, 4, and 6) were separated by 10% SDS-PAGE and visualized using Coomassie
brilliant blue staining (lanes 1 and 2), a phosphor imager tritium
screen exposed for 5 d (lanes 3 and 4), and a western blot with
eEF-1A antibody (lanes 5 and 6). Equal protein (1 µg total) was used
per lane for the 40,000g supernatant and purified eEF-1A. Molecular mass standards are shown at left (in kD). The experiment was repeated multiple times. The arrow indicates the migration of the 50-kD standard.
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[14C]Ethanolamine-labeled peptides were
compared in Arabidopsis and several cell culture lines of carrot,
tobacco, and maize endosperm cells. When
[14C]ethanolamine-labeled proteins from
Arabidopsis seedlings were analyzed, several radiolabeled bands could
be detected (Fig. 2A). The predominant
[14C]ethanolamine band on a gel was 49 kD and
cross-reacted with antibodies to eEF-1A (data not shown).
[14C]Ethanolamine was incorporated equally into
soluble and microsomal peptides in the root as well as the shoot and
leaf fractions. Little or no labeled protein was recovered from rapidly
growing carrot cells (5 g harvested/4 d in culture) or from maize
endosperm cultures (data not shown). With tobacco cells grown in
suspension culture a [14C]ethanolamine-labeled
49-kD peptide was observed on an autoradiograph of a western blot (Fig.
2B).
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| Figure 2.
Arabidopsis seedlings and tobacco cells grown in
suspension culture were incubated with [14C]ethanolamine
for 2 d and harvested as described in ``Materials and Methods''.
A, Arabidopsis protein. Autoradiograph of 10% SDS-PAGE of
40,000g pellet (microsomes) (lanes 1 and 2) and
supernatant (lanes 3 and 4) fractions (26 µg of total protein per
lane). Lanes 1 and 3, Roots; lanes 2 and 4, leaves and shoots. B,
Tobacco protein. Western blot using eEF-1A antibodies (lanes 1 and 2)
and autoradiograph of the western blot exposed to film for 1 month
(lanes 3 and 4). Lanes 1 and 3, 40,000g supernatant;
lanes 2 and 4, 40,000g microsomes. The cross-reacting polypeptides below 49 kD are typical of proteolytic breakdown products.
Molecular mass standards are shown at left (in kD).
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The most efficient labeling of eEF-1A was found in two slowly growing
carrot cell lines. A newly isolated embryogenic cell line, which
contained large (1 mm) clusters of cells (10-20 µm in diameter) that
were lipid rich based on the fact that a layer of fat was collected
during membrane isolation, consistently yielded a high specific
activity of purified [14C]et-eEF-1A
(50,000 ± 400 dpm/mg). The other culture that had a high specific
activity of purified [14C]et-eEF-1A
(70,000 ± 8000 dpm/mg) was a slowly growing cell line derived
from embryogenic cultures, which had been in culture for over 10 years
(Chen and Boss, 1990). The cells appeared as a fine suspension and
yielded 0.4 g fresh weight after 4 d in culture. Because
eEF-1A was the only protein that incorporated ethanolamine in the
carrot cell culture system (Fig. 1), and because the specific activity
was highest in the slower-growing cell lines, we used the carrot
cells to characterize this posttranslational modification.
To determine the best time during the cell growth cycle for
incorporation of [14C]ethanolamine, carrot
cells were labeled for 2-d periods at various times during the cell's
7-d growth cycle and harvested to obtain a 40,000g
supernatant and membrane fractions, as described in ``Materials and Methods''. The amount of [14C]ethanolamine
incorporated into the 49-kD peptide increased over the growth cycle of
the culture (Fig. 3).
[14C]et-eEF-1A was found in the soluble,
endomembrane, and plasma membrane fractions. The specific activity of
[14C]et-eEF-1A increased with time of
incubation and/or increased concentration of isotope per gram fresh
weight of cells. We routinely obtained good incorporation if we
incubated log-phase cells for 2 d with 10 µCi
[14C]ethanolamine/g fresh weight.
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| Figure 3.
Carrot cells were incubated with
[14C]ethanolamine for 2 d during different times of
the growth cycle. Cells were weighed, homogenized, and centrifuged to
obtain a 40,000g supernatant, endomembrane, and plasma
membrane fraction. The final specific activity recovered was 30 µCi
[14C]ethanolamine/g fresh weight for cells labeled from d
1 to 3, 25 µCi/g fresh weight for cells labeled from d 2 to 5, and 22 µCi/g fresh weight for cells labeled from d 5 to 7. Shown is an autoradiograph of 10% SDS-PAGE with 40 µg of total protein/lane for
supernatant and endomembrane fractions, 10 µg of total protein for
the plasma membrane fraction. Lanes 1 to 3, 40,000g
supernatant from cells labeled from d 1 to 3, 2 to 5, and 5 to 7, respectively; lanes 4 to 6, endomembranes; and lanes 7 to 9, plasma
membrane of cells from the same respective culture times. The
experiment was repeated twice with duplicate samples. A representative
experiment is shown. The arrow indicates the migration of the 50-kD
standard.
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The [14C]Ethanolamine Is Part of a PGE
Posttranslational Modification
To determine whether [14C]ethanolamine
incorporated into eEF-1A is part of the PGE modification, as previously
reported in animal cells (Dever et al., 1989; Rosenberry et al., 1989;
Whiteheart et al., 1989), or whether it is part of a GPI anchor, cells
were incubated with [3H]glycerol,
[32P]Pi, [14C]myristic
acid, [3H]palmitoleic acid,
[14C]linoleic acid, or
[3H]inositol, and the incorporation into eEF-1A
was compared with that of [14C]ethanolamine.
For these experiments, eEF-1A was purified from the 40,000g
soluble-protein fraction, and the purified protein was precipitated
with TCA, washed with acetone to remove all noncovalently bound lipids,
and counted in a scintillation counter or analyzed by SDS-PAGE and
autoradiography. Analysis of the protein by SDS-PAGE and
autoradiography indicated comigration of
[14C]ethanolamine,
[14C]myristic acid,
[14C]linoleic acid, and
[32P]Pi with eEF-1A in both the purified and
the 40,000g soluble fractions (Fig.
4). When the amount of
[14C]ethanolamine and
[3H]glycerol was quantitated per
milligram of delipidated protein, there was an increase in
incorporation in the column-purified protein compared with the soluble
fraction (Table II). This means that
eEF-1A was the only or primary protein incorporating these isotopes.
The potential for nonspecific lipid binding to the TCA precipitate was
tested by incubating [14C]PC with
nonradiolabeled protein prior to TCA precipitation. Less than 1% of
the total PC added was recovered after TCA precipitation, indicating
that the extraction was effective and that noncovalently bound lipids
were removed (Table I).
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| Figure 4.
Carrot cells (d 2) were incubated with
[14C]ethanolamine (7.5 µCi/g fresh weight),
[14C]myristic acid (25 µCi/g fresh weight),
[3H]inositol (50 µCi/g fresh weight), or with
[14C]linoleic acid 0.1 mCi/g fresh weight) for 2 d.
Cells were placed into phosphate-free medium 30 min before the addition
of [32P]Pi (0.4 mCi/g fresh weight) and incubated with
isotope for 16 h. Cells were harvested as described in
``Materials and Methods''. A, Proteins were separated by 10%
SDS-PAGE and visualized by Coomassie brilliant blue staining. Lanes 1 to 3, Partially purified eEF-1A fraction (2 µg of total protein);
lanes 4 to 6, microsomal fraction (20 µg of total protein); and lanes
7 to 9, supernatant fraction (20 µg of total protein). In lanes 1, 4, and 7, cells were labeled with [14C]ethanolamine; in
lanes 2, 5, and 8 with [14C]myristic acid; and in lanes
3, 6, and 9 with [3H]inositol. B, Image of gel shown in A
using a phosphor imager tritium screen exposed for 13 d. C,
Purified eEF-1A labeled with [32P]Pi. Lane 1, 10%
SDS-PAGE visualized by Coomassie brilliant blue staining (2 µg); lane
2, autoradiograph of [32P]Pi eEF-1A exposed to film for
5 d. D, [14C]Linoleic acid-labeled cells:
40,000g supernatant protein and microsomes. Lanes 1 and
2, 10% SDS-PAGE visualized by Coomassie brilliant blue staining. Lane
1, Microsomes (20 µg of total protein); lane 2, soluble protein (20 µg of total protein); and lanes 3 and 4, autoradiograph of SDS-PAGE.
Arrows indicate migration of 50-kD standard. Molecular mass standards
are shown at left (in kD). In vivo-labeling experiments were repeated
at least twice with duplicates.
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View this table:
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Table II.
Components of the carrot eEF-1A PGE
posttranslational modification
Carrot cells were labeled with [14C]ethanolamine,
[14C]myristic acid, and [3H]glycerol as
described in ``Materials and Methods''. Supernatant
(40,000g), microsomal pellet, and purified eEF-1A fractions
were TCA precipitated and delipidated. The numbers for the delipidated
protein represent the average of three samples.
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When [14C]myristic acid was added to the cells,
the specific activity of eEF-1A did not increase with purification. In
fact, purified eEF-1A had a lower specific activity than the mixture of
myristoylated proteins in the soluble fraction (Table II). This would
be anticipated if myristate entered the fatty acid biosynthesis pathway
before attachment to eEF-1A. For example, myristate could form a CoA
intermediate, be converted to palmitic or linoleic acid, and then
be incorporated as one of two fatty acids on the PGE glycerol backbone.
Like myristate, the incorporation of
[14C]linoleic acid is consistent with the
presence of fatty acids bound via a phospholipid. To test for
nonspecific binding of fatty acids, carrot cells were incubated with
[3H]palmitoleic acid, a fatty acid only found
in a rare lipid modification of proteins (Casey et al., 1994), and the
incorporation into purified, delipidated eEF-1A was monitored. Very
little incorporation was found (3 dpm above background) even when the
cells were incubated with 35 µCi/g fresh weight.
Because inositol phosphates and other inositol metabolites that may
coelute with eEF-1A on the mini-columns would precipitate with TCA (Cho
et al., 1995), we could not use this method to assess the presence of
inositol or phosphate in purified eEF-1A. We could never detect
[3H]inositol comigrating with eEF-1A by
SDS-PAGE and autoradiography. On one occasion, after a 6-month
exposure, a very faint band was detected on an autoradiograph of a
western blot with [3H] inositol-labeled
partially purified eEF-1A (data not shown). This may have resulted from
noncovalently bound phosphatidylinositol, which migrates in the same
region on SDS-PAGE (I. Brglez and W.F. Boss, unpublished results).
The incorporation of ethanolamine, glycerol, and phosphate is
consistent with a PGE modification. Incorporation of
[14C]myristic and
[14C]linoleic acid (Fig. 4, B and D) suggests
the attachment of one or two fatty acids to the glycerol backbone of
the PGE modification. Evidence for the presence of a complete
phospholipid moiety, PE, covalently attached to eEF-1A has only been
reported in Chinese hamster fibroblast cells (Hayashi et al., 1989). In
rabbit myeloma and human T-lymphocyte cells, incorporation of fatty
acid into eEF-1A was not reported. With the carrot eEF-1A, the
incorporation of [14C]myristic acid into the
purified, delipidated protein and comigration of
[14C]myristic acid and
[14C]linoleic acid with eEF-1A on SDS-PAGE
suggests that the entire phospholipid is present.
Because the incorporation of [32P] into eEF-1A
could also result from phosphorylation of amino acids (Venema et al.,
1991; Yang et al., 1993), and because even a low percentage of
radiolabeled lipid adhering to eEF-1A could potentially affect the
interpretation of the data, we attempted to hydrolyze the phospholipid
moiety and analyze it by TLC. However, after delipidation and
hydrolysis of the protein with 6 N HCl for 16 h at
110°C we could not recover a significant amount of the released PGE
product for further analysis. Enzymatic hydrolysis of the proteolipid
also did not yield sufficient lipid for analysis. For these reasons, we
used MS analysis to confirm the presence of a covalently bound PGE
modification.
For the MS studies [14C]et-eEF-1A and
nonradiolabeled carrot eEF-1A were purified, separated by SDS-PAGE, and
digested with trypsin as described in ``Materials and Methods''. The
tryptic peptides were separated by reverse-phase HPLC, as described in
``Materials and Methods''. HPLC fractions containing the
[14C]ethanolamine peptides were identified by
counting an aliquot of each fraction in Scintiverse II cocktail in a
scintillation counter and resulted in a profile identical to that
obtained by Whiteheart et al. (1989; data not shown). The major
radioactive peaks were combined into three fractions and analyzed by
MALDI-MS. Similar spectra were obtained for fractions 2 and 3. Two
signals at m/z 2721 and 2737, representing intact molecule
plus one proton, were identified as corresponding to the tryptic
peptide containing amino acid residues 279-301 with one PGE molecule
attached (Fig. 5). The PGE moiety adds
197 mass units to the peptide, which has an intact molecule plus one
proton at m/z 2524. The amino acid sequence for the tryptic
peptide spanning residues 279 to 301 is
SVEMHHEALQEALPGDNVGFNVK. The predicted PGE attachment site is glutamate no. 285 (shown in italic; Dever et al., 1989; Rosenberry et al., 1989; Whiteheart et al., 1989). The second signal at
m/z 2737 corresponds to the same peptide where Met oxidation
took place, adding an additional 16 mass units to the peptide.
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| Figure 5.
MS analysis of [14C]et-eEF-1A
tryptic peptides. MALDI-MS of concentrated HPLC fractions from two
peaks of the [14C]ethanolamine HPLC profile. A
signal at m/z 2721 was detected, which corresponds to
the predicted mass of tryptic peptide amino acids 279 to 301 covalently
bound to one PGE molecule. The signal for peptide 279 to 301 plus PGE
moiety was detected from two separate sample preparations.
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Conservation of the PGE Attachment Site
A comparison of the amino acid sequences of eEF-1A from other
organisms is shown in Table III. Two
attachment sites for the PGE posttranslational modification have been
identified in rabbit, mouse, and human (analogous to Glu residues 285 and 362), as determined by amino acid sequencing and
fast-atom-bombardment MS analysis of eEF-1A tryptic peptides (Dever et
al., 1989; Rosenberry et al., 1989; Whiteheart et al., 1989). These two
sites are highly conserved in plants with sequence motifs of
SVEMHHEA/SLL/QEALPGDNVGFNVK (amino acids 279-301) and
KFAEXXK (amino acids 282-288). It is interesting that no
ethanolamine labeling has been reported in yeast, and yeast do not have
a glutamate residue at position 299 (285 in plants) or the
KFAEXXK motif of the second attachment site (amino acids
361-369) (Table III; Cavallius et al., 1993).
What Is the Distribution of PGE eEF-1A?
To gain some insight into the possible subcellular distribution of
the PGE posttranslational modification of eEF-1A, we wondered if
[14C]et-eEF-1A was preferentially associated
with membranes or the cytoskeleton. The in vivo-labeling studies of
carrot cells using [14C]ethanolamine indicated
the presence of [14C]et-eEF-1A in the soluble,
endomembrane, and plasma membrane fractions of the cell (Fig. 3). The
fatty acids on the PGE modification could form a lipid anchor holding
eEF-1A to the membranes and cytoskeleton or lipid-associated proteins.
Under the homogenizing conditions optimized for purifying soluble
protein, most of the eEF-1A was soluble. However, if a homogenizing
buffer was used that stabilizes actin filaments (Abe et al., 1992),
most (>90%) of the [14C]et-eEF-1A was
recovered in the microsomes (Fig. 6).
These data suggest that [14C]et-eEF-1A is
capable of binding actin.
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| Figure 6.
The recovery of [14C]et-eEF-1A in
the 40,000g microsomal pellet and supernatant was
compared using cytoskeleton isolation buffer or regular buffer as
described in ``Materials and Methods''. A, 40,000g
supernatant and microsomal pellet fractions from two different
homogenizing buffers separated on 10% SDS-PAGE and visualized with
Coomassie brilliant blue staining. Lanes 1and 2, Homogenizing with
regular buffer: lane 1, supernatant; lane 2, microsomes. Lanes 3 and 4, Homogenizing with cytoskeleton isolation buffer: lane 3, supernatant;
lane 4, microsomes. B, Image of the gel in A using a phosphor imager tritium screen exposed for 4 d. The experiment was repeated two times with duplicates. The arrow indicates the migration of the 50-kD
standard.
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To test F-actin binding, [14C]et-eEF-1A was
incubated with different concentrations of actin under actin
sedimentation conditions. The supernatant (G-actin) and pellet
(F-actin) fractions were analyzed by SDS-PAGE and visualized by
staining with Coomassie brilliant blue (Fig.
7) and with a phosphor imager tritium
screen. The relative distribution of Coomassie blue-stained protein and [14C]ethanolamine was similar. These data
indicate that [14C]et-eEF-1A, like eEF-1A, will
bind F-actin, but they do not show preferential actin binding by the
[14C]et-eEF-1A.
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| Figure 7.
Different concentrations of actin and
[14C]et-eEF-1A were mixed together in sedimentation
buffer for an actin-binding assay as described in ``Materials and Methods''. The mixture was separated at 15,000 rpm into supernatant
and pellet fractions by centrifugation. A, The supernatant (s) and
pellet (p) fractions were analyzed by 12% SDS-PAGE and visualized with
Coomassie brilliant blue staining. Lanes 1 and 2, Actin alone (40 µg); lanes 3 and 4, eEF-1A alone (10 µg); lanes 5 and 6, BSA alone
(10 µg); lanes 7 and 8, actin (40 µg) plus eEF-1A; lanes 9 and 10, actin (20 µg) plus eEF-1A; lanes 11 and 12 actin (40 µg) plus BSA;
and lanes 13 and 14, actin alone (20 µg). B, Image of the gel in A
using a phosphor imager tritium screen exposed for 4 weeks. The
experiment was repeated three times with duplicates. A representative
experiment is shown. The arrow indicates the migration of the 50-kD
standard.
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Triton X-114 Partitioning of eEF-1A
Triton X-114 is a detergent used to separate hydrophilic proteins
from amphipathic integral membrane proteins in a temperature-dependent phase separation (Bordier, 1981). Hydrophilic proteins (>80%) from
lysed cell extracts and isolated membranes or purified proteins without
a lipid anchor remain in the aqueous phase, whereas amphipathic integral membrane and lipid-anchored proteins (>80%) enter the detergent-rich phase after separation (Hooper, 1992). Degradation or
cleavage of the protein's lipid anchor will shift partitioning of the
protein from the detergent-rich to the detergent-poor phase. Previous
work showed that [3H]ethanolamine-labeled
eEF-1A from mouse cell lysates partitioned into the aqueous phase
(Tisdale and Tartakoff, 1988). To determine the effect of the PGE
attachment on carrot eEF-1A Triton X-114 partitioning, eEF-1A labeled
with [14C]ethanolamine and
[14C]myristic acid was mixed with Triton X-114,
and the aqueous and detergent-rich phases were analyzed by SDS-PAGE and
exposed to a phosphor imager tritium screen for 11 d. Purified
[14C]et-eEF-1A partitioned equally between the
aqueous and the detergent-rich phases. The distribution of
[14C]myristic acid-labeled purified eEF-1A
analyzed by the phosphor imager was consistent with distribution of the
Coomassie blue-stained protein. The identical patterns indicate that
there was no preference for the
[14C]myristoylated isoform to partition into
the detergent phase (Fig. 8). Crude
eEF-1A from a 40,000g soluble fraction of
[14C]ethanolamine-labeled and
[14C]myristic acid-labeled cells also
partitioned mostly into the aqueous phase. Even when the eEF-1A in the
40,000g microsomal pellet was used, both
[14C]et-eEF-1A and
[14C]myristic acid-labeled eEF-1A were observed
in the aqueous phase. In a separate experiment, reEF-1A from tomato
expressed in Escherichia coli with a 6× His tag partitioned
equally between the aqueous and detergent-rich phases (Fig. 8C). The
increase in the percentage of protein partitioning into the
detergent-rich phase with the E. coli-expressed protein,
which would not have a PGE modification, probably resulted from an
increase in the percentage of denatured protein. Importantly, the
reEF-1A was active in an actin-binding assay and, when phosphorylated,
activated PI 4-kinase, indicating that the PGE modification was not
essential for either actin binding or for PI 4-kinase activation (data
not shown).
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| Figure 8.
Purified [14C]et-eEF-1A, purified
[14C]myristate eEF-1A, 40,000g microsomal
proteins, BSA, and tomato reEF-1A (15 µg) were partitioned into the
aqueous (a) and detergent-rich (d) phase as described in ``Materials and Methods''. A, Aqueous and detergent-rich fractions were separated
by 10% SDS-PAGE and visualized by Coomassie brilliant blue staining. Lanes 1 and 2, [14C]et-eEF-1A; lanes 3 and 4, [14C]myristic acid eEF-1A; lanes 5 and 6, [14C]myristic acid-labeled 40,000g
microsomal pellet; lanes 7 and 8, [14C]ethanolamine-labeled 40,000g
supernatant; lanes 9 and 10, [14C]myristic acid-labeled
40,000g supernatant; and lanes 11 and 12, BSA. B, Image
of the gel in A using a tritium phosphor imager screen exposed for 4 weeks. C, Tomato reEF-1A visualized by staining with Coomassie
brilliant blue staining. Lane 1, Aqueous phase; and lane 2, detergent-rich phase. The experiment was repeated two times with
duplicates. A representative experiment is shown. The arrow indicates
the migration of the 50-kD standard.
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BSA was used as a positive control for aqueous partitioning. Greater
than 90% of the BSA was observed in the aqueous phase as visualized by
SDS-PAGE stained with Coomassie blue (Fig. 8A). If the PGE had a
significant effect on hydrophobicity,
[14C]et-eEF-1A and
[14C]myristic acid-labeled eEF-1A would have
gone preferentially into the detergent-rich phase relative to the
amount of protein. There was no evidence of preferential partitioning
based on Coomassie blue staining that visualized the total protein and
analysis of [14C]et-EF-1A and
[14C]myristoylated-eEF-1A by the phosphor
imager. Clearly, the PGE modification is not the limiting factor in how
eEF-1A partitions.
| |
DISCUSSION |
We have shown that [14C]ethanolamine is
incorporated into a 49-kD polypeptide in both Arabidopsis and carrot
cells grown in suspension culture. In the carrot cells the 49-kD
peptide is the only ethanolamine-labeled protein recovered. The
[14C]ethanolamine-labeled protein copurified
with eEF-1A and cross-reacted with eEF-1A antibodies. Because of
previous reports of ethanolamine-labeled and GPI-anchored proteins in
plants (Morita et al., 1996; Takos et al., 1997), it was essential to
use MS to analyze the composition of the modification. MALDI-MS
analysis of tryptic peptides revealed that the purified carrot eEF-1A
has a PGE posttranslational modification similar to that reported for
rabbit and human eEF-1A (Dever et al., 1989; Rosenberry et al., 1989;
Whiteheart et al., 1989). The MALDI-MS data are consistent with the in
vivo-labeling data. In addition, the in vivo-labeling studies but not
the MALDI-MS data provide evidence for the presence of long-chain fatty
acids on the glycerol backbone of the molecule. Both
[14C]myristate and
[14C]linoleic acid-labeled polypeptides
comigrated with eEF-1A in SDS-PAGE and copurified with the delipidated
protein. These data are consistent with myristate first being
elongated, forming a CoA intermediate, and then being incorporated into
the glycerol backbone of the phospholipid.
[14C]Linoleic acid was incorporated into
several proteins, including eEF-1A. The incorporation of
[14C]linoleic acid is less than that of
[14C]myristate. This is typical for the
incorporation of exogenously added fatty acid into phospholipids and
reflects the decreased synthesis of the fatty acyl CoA intermediate by
the longer-chain fatty acid. eEF-1A does not contain the typical
MGXXXSXX motif considered necessary for N-myristoylation
(Johnson et al., 1994). These data, in addition to the observed
decreased specific activity of [14C]myristic
acid eEF-1A, indicate that myristic acid was not directly bound to the
protein but rather entered fatty acid or phospholipid metabolic
pathways prior to binding eEF-1A. Previous MS data and labeling studies
have demonstrated that rat, rabbit, and human eEF-1A have a PGE group
covalently linked via an amide bond to Glu through the ethanolamine
nitrogen (Dever et al., 1989; Rosenberry et al., 1989; Whiteheart et
al., 1989). Our data indicate that a similar PGE modification to a
peptide containing this conserved Glu consensus sequence is present in
carrot eEF-1A. The fatty acid ester bond might not have been
stable during sample preparation. Alternatively, PE modified peptides
(incorporating fatty acyl groups) are not detected because they may
have a significantly lower response in MALDI-MS analysis relative to
the PGE-modified and unmodified peptides present in the HPLC fractions.
However, in vivo-labeling studies confirm the presence of covalently
bound fatty acids forming a phospholipid attachment. This type of
covalently bound phospholipid could facilitate association of the
protein with membrane lipids and hydrophobic proteins in vivo.
Triton X-114 partitioning of both
[14C]ethanolamine and
[14C]myristic acid eEF-1A did not indicate a
clear preference of the lipid-modified proteins for the detergent-rich
phase. GPI-anchored proteins, which contain several sugars in addition
to PI, preferentially partition to the detergent-rich phase (Hooper,
1992), whereas myristoylated and PGE-modified proteins partition to the
aqueous phase (Tisdale and Tartakoff, 1988). Our data indicate that the additional fatty acids on the PGE moiety were not sufficient to enhance
partitioning to the detergent-rich phase. The fact that a greater
percentage of the E. coli-expressed recombinant protein partitioned into the detergent-rich phase indicated that partitioning correlated with the increase in the percentage of denatured protein and
not the PGE modification.
Because eEF-1A is found widely distributed throughout the cell, it is
hypothesized that its function and or location may be regulated by
different posttranslational modifications. However, except for a
requirement for phosphorylation for activation of PI 4-kinase (Yang and
Boss, 1994), the effects of posttranslational modifications of eEF-1A
have not been demonstrated either in vivo (Cavallius et al., 1997) or
in vitro (Venema et al., 1991). We have shown that the PGE
modification alone does not appear to be a limiting factor for either
actin binding or Triton X-114 partitioning, and that the PGE
modification is not essential for PI-4 kinase activation, because
phosphorylated reEF-1A, which has no PGE, will activate PI-4 kinase. It
may be that the posttranslational modifications, PGE, phosphorylation,
and methylation, work in combination to influence the translocation and
function of eEF-1A within the cell. We are currently using
site-directed mutagenesis of the PGE attachment to study the roles of
this posttranslational modification alone and in combination with
phosphorylation sites.
| |
FOOTNOTES |
1
This research was supported by the National
Science Foundation (grant no. MCB-9604285 to W.F.B.) and by a Patricia
Roberts Harris fellowship to W.D.R. Acquisition of mass spectral data at Michigan State University-National Institutes of Health (NIH) Mass
Spectrometry Facility was supported in part by the NIH (grant no.
RR00480).
*
Corresponding author; e-mail wendy_boss{at}ncsu.edu; fax
1-919-515-3436.
Received January 8, 1998;
accepted April 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
[14C]et-eEF-1A, [14C]ethanolamine-labeled eEF-1A.
eEF-1A, eukaryotic elongation factor 1.
GPI, glycosylphosphatidylinositol.
MALDI-MS, matrix-assisted laser desorption/ionization-MS.
PC, phosphatidylcholine.
PE, phosphatidylethanolamine.
PGE, phosphoglycerylethanolamine.
PI-4 kinase, phosphatidylinositol-4
kinase.
reEF-1A, recombinant eEF-1A.
TFA, trifluoroacetic acid.
| |
ACKNOWLEDGMENTS |
We would like to thank Drs. Steve Huber and Marcus Bachman
(North Carolina State University, Raleigh) for use of the HPLC and
instruction on purification of peptides; Dr. Leo Parks (North Carolina
State University, Raleigh) for the gift of the
[3H]palmitoleic acid; Drs. Brian Edmonds and
John S. Condeelis (Albert Einstein College of Medicine, Bronx, NY) for
the antibody to D. discoideum eEF-1A (ABP-50); and Dr.
Christine K. Shewmaker (Calgene, Inc., Davis, CA) for the tomato eEF-1A
clone.
| |
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Abstract
Introduction
