A hydrophilic cation-binding protein of
Arabidopsis thaliana, AtPCaP1, is localized to plasma
membrane via N-myristoylation and interacts with
calmodulin and the phosphatidylinositol phosphates
PtdIns(3,4,5)P
3
and PtdIns(3,5)P
2
Nahoko Nagasaki, Rie Tomioka and Masayoshi Maeshima
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan
The intracellular localization of proteins is critical for
expression of their cellular function, and is determined
by several mechanisms, including their primary
sequences, post-translational processing, covalent mod-
ifications and affinity to other elements. Most soluble
proteins are localized to the cytoplasm, intra-organelle
spaces, cytoskeletons or secreted out of the cells. How-
ever, some parts of hydrophilic proteins in cells can be
Keywords
Arabidopsis; calcium; myristoylation;
phosphatidylinositol phosphate; plasma
membrane
Correspondence
M. Maeshima, Laboratory of Cell Dynamics,
Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan
Fax: +81 52 789 4096
Tel: +81 52 789 4096
E-mail: maeshima@agr.nagoya-u.ac.jp
(Received 19 October 2007, revised 5
February 2008, accepted 5 March 2008)
doi:10.1111/j.1742-4658.2008.06379.x
A hydrophilic cation-binding protein, PCaP1, was found to be stably
bound to the plasma membrane in Arabidopsis thaliana. PCaP1 was quanti-
fied to account for 0.03–0.08% of the crude membrane fractions from roots
and shoots. Its homologous protein was detected in several plant species.
We investigated the mechanism of membrane association of PCaP1 by
transient expression of fusion protein with green fluorescent protein. The
amino-terminal sequence of 27 residues of PCaP1 had a potential to local-
ize the fusion protein with green fluorescent protein to the plasma mem-
brane, and the substitution of Gly at position 2 with Ala resulted in the
cytoplasmic localization of PCaP1. When PCaP1 was expressed in the
in vitro transcription ⁄translation system with [
3
H]myristic acid, the label
was incorporated into PCaP1, but not into a mutant PCaP1 with Gly2
replaced by Ala. These results indicate that PCaP1 tightly binds to the
plasma membrane via N-myristoylation at Gly2. We examined the binding
capacity with phosphatidylinositol phosphates (PtdInsPs), and found that
PCaP1 selectively interacts with phosphatidylinositol 3,5-bisphosphate and
phosphatidylinositol 3,4,5-triphosphate. Competition assay with the N-ter-
minal peptide and mutational analysis revealed that PCaP1 interacts with
these two PtdInsPs at the N-terminal part. Interaction of PCaP1 with the
membrane and PtdInsPs was not altered in the presence of Ca
2+
at physio-
logical concentrations. Furthermore, calmodulin associated with PCaP1 in
aCa
2+
-dependent manner, and its association weakened the interaction of
PCaP1 with PtdInsPs. These results indicate that the N-terminal part is
essential for both N-myristoylation and interaction with PtdInsPs, and that
PCaP1 may be involved in intracellular signalling through interaction with
PtdInsPs and calmodulin.
Abbreviations
CaM, calmodulin; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; MAP, methionine aminopeptidase; NMT, myristoyl-
CoA:protein N-myristoyltransferase; PCaP1, plasma membrane-associated cation-binding protein; PtdIns(3,4,5)P
3
, phosphatidylinositol
3,4,5-triphosphate; PtdIns(3,5)P
2
, phosphatidylinositol 3,5-bisphosphate; PtdInsP, phosphatidylinositol phosphate.
FEBS Journal 275 (2008) 2267–2282 ª2008 The Authors Journal compilation ª2008 FEBS 2267
associated with the plasma and organelle membranes
via covalent modification with lipids, formation of
complexes with membrane integral proteins and associ-
ation with membrane components such as membrane
lipids. The strength and reversibility of the association
with membranes depends on the biochemical proper-
ties of the proteins.
Covalent modifications with lipids, in particular,
are of interest in relation to the cell signalling and
regulatory functions through these proteins [1–5].
Lipid modifications, in combination with other post-
translational changes, some reversible, often cause
proteins to undergo extensive intracellular transloca-
tion. Four types of lipid modification are known:
N-myristoylation, prenylation, palmitoylation and
modification with glycosylphosphatidylinositol (GPI)
anchor proteins [5]. Palmitoylation is the process of
introduction of palmitic acid into protein by substitu-
tion for a hydrogen atom of a Cys residue (S-acyla-
tion). Typical proteins with palmitoylation are
a-subunits of heterotrimeric G-proteins [6]. Palmitoy-
lation of proteins is a reversible process in living cells.
Therefore, the intracellular localization and physiolog-
ical functions can be regulated in cells. N-myristoyla-
tion is the covalent attachment of a myristoyl group
via an amide bond to the N-terminal Gly residue of
a nascent polypeptide. For example, some a-subunits
of G-protein heterotrimers, some small G-proteins
and several non-receptor-type tyrosine kinases are
N-myristoylated proteins. Proteins with lipid modifica-
tions come in many shapes, sizes and functions, even
in plants [7]. Specific primary sequences, such as a
myristoylation signal motif, determine the type of
lipid modification.
In addition to covalent lipid modifications, the spe-
cific interaction with phosphatidylinositol phosphates
(PtdInsPs) in the membrane plays a critical role in the
regulation of the function and intracellular localization
of proteins [8–11].
Very recently, a novel hydrophilic cation-binding
protein was identified in Arabidopsis thaliana [12]. The
protein is composed of 225 amino acid residues and is
rich in Glu and Lys. The protein has no transmem-
brane domain, but is associated with the plasma mem-
brane, and was tentatively named AtPCaP1 (hereafter
referred to as PCaP1). The gene coding for PCaP1 was
constitutively expressed in most organs, and the
mRNA level was enhanced by the treatment with a
pathological elicitor, sorbitol, and copper [12]. How-
ever, the physiological function of PCaP1 is unclear.
In this study, we focused our attention on the bio-
chemical mechanism of the association of PCaP1 with
the plasma membrane. We found that the protein con-
tains a candidate for the myristoylation signal at the
N-terminal region, and investigated this. Biochemical
analyses, including in vitro myristoylation, demon-
strated the N-myristoylation of PCaP1. In addition,
PCaP1 has a candidate for association with PtdInsPs.
We examined this possibility and determined quantita-
tively the specificity of the PtdInsPspecies. Further-
more, we observed that PCaP1 associated with
calmodulin (CaM) in the presence of calcium. These
observations are essential for understanding the bio-
chemical roles of the novel cation-binding protein and
its related proteins in various organisms. The present
study revealed that PCaP1 is a unique protein, which
is N-myristoylated and associated with specific
PtdInsPs. The biochemical meaning of these properties
is discussed.
Results
Immunochemical detection of PCaP1 orthologues
in several plant species
PCaP1 is composed of 225 amino acids and is rich
in Glu (44 residues), Lys (35 residues) and Val (25
residues). The protein has characteristic repeats
(IEEKK, VEEKK and VEETKK) (Fig. 1A). To
date, no motif has been found for enzymatic
function. A possible candidate for N-myristoylation
exists at the N-terminal region, as described later.
PCaP1 has many Ser and Thr residues, and some
residues have been estimated to be phosphorylation
sites. A homologous protein with high identity with
PCaP1 was found in Nicotiana tabacum by blast
search (http://blast.ddbj.nig.ac.jp/top-j.html) (Fig. 1A).
This protein was named DREPP1 (developmentally
regulated plasma membrane protein) [13]. Although
the protein was detected in the plasma membrane
and endomembrane fractions, its physiological and
biochemical properties are unknown. The N-terminal
halves are highly conserved between the two
sequences, suggesting that PCaP1 and its orthologues
are not unique to A. thaliana.
The calculated molecular mass of PCaP1 is 24 584;
however, the protein was detected with a molecular
mass of 36 kDa in an immunoblot with anti-PCaP1
IgG (Fig. 1B), which was raised against the peptide
with internal sequence of PCaP1 (positions 152–166).
The difference between the calculated and apparent
size may be caused by the amount of dodecyl-sulfate
bound to PCaP1 and ⁄or the structure in SDS.
Immunoblotting showed bands in Raphanus sativus
(radish, 41 kDa), Brassica rapa (turnip, 42 kDa),
Brassica rapa var. glabra Regel (Chinese cabbage,
A novel cation-binding myristoylated protein N. Nagasaki et al.
2268 FEBS Journal 275 (2008) 2267–2282 ª2008 The Authors Journal compilation ª2008 FEBS
43 kDa) and Brassica oleracea var. italica (broccoli,
41 kDa) (Fig. 1B). The immunostained bands disap-
peared when the corresponding peptide was added to
the reaction medium. Thus, these bands were ortho-
logues of PCaP1. The low intensity of immunostain-
ing, except for A. thaliana, may be caused by the
partial difference in the sequence corresponding to
the epitope. We did not examine the membrane
preparation from N. tabacum, because the corre-
sponding sequence is not a match with that of
PCaP1 (Fig. 1A).
Quantification of PCaP1 in the membrane and
soluble fractions
To determine the amount of PCaP1 in tissues and the
distribution of PCaP1 in the membrane and soluble
fractions (by an immunochemical method), we pre-
pared the recombinant PCaP1 as the standard protein.
As shown in Fig. 2A, a highly purified preparation of
PCaP1 without any tag was obtained. The protein was
analysed by SDS-PAGE and immunoblotting with an
anti-PCaP1 IgG to obtain a calibration curve
A
B
Fig. 1. Detection of PCaP1 orthologues in plants. (A) Amino acid sequence alignment of A. thaliana PCaP1 and N. tabacum DREPP1.
Identical (*) and conserved (:) residues are marked. Gaps introduced to maximize alignment scores are denoted by hyphens. A putative
N-myristoylation site of PCaP1 is underlined. The overlined sequence was used for preparation of the anti-PCaP1 IgG. Characteristic VEEKK
motifs and variants are boxed. Possible phosphorylation sites were predicted using the NETPHOS 2.0 program (http://www.cbs.dtu.dk/
services/NetPhos/). Open circles indicate possible phosphorylation residues with a high score of more than 0.8, and filled circles indicate the
target residues of protein kinase-C-like enzymes with a high score of more than 0.7. (B) Immunoblot detection of PCaP1 orthologous protein
in crude membrane fractions with anti-PCaP1. Lanes 1 and 6, A. thaliana; lanes 2 and 7, Raphanus sativus; lanes 3 and 8, Brassica rapa;
lanes 4 and 9, B. rapa var. glabra; lanes 5 and 10, B. oleracea var. italica. The amount of protein applied was 4 lg for A. thaliana and 40 lg
for the other plants.
N. Nagasaki et al. A novel cation-binding myristoylated protein
FEBS Journal 275 (2008) 2267–2282 ª2008 The Authors Journal compilation ª2008 FEBS 2269
(Fig. 2B,C). The crude membrane fractions and soluble
fractions were prepared from shoots and roots and
subjected to immunoblotting (Fig. 2D). The absolute
amount of PCaP1 was calculated using a standard
curve. Most PCaP1 was recovered in the membrane
fractions, and the PCaP1 amounts in the shoot and
root fractions were 0.79 and 0.29 lgÆmg
)1
of total
membrane protein, respectively. There was only a trace
amount of PCaP1 in the soluble fractions. Thus,
PCaP1 was predominantly localized to the membrane
in the tissues, and was present at 0.079% and 0.029%
of total membrane proteins in the shoots and roots,
respectively.
The stability of the interaction of PCaP1 with the
plasma membrane was examined by treating the mem-
branes with several reagents (Fig. 3). PCaP1 was not
released from the plasma membrane by treatment with
0.1 mNaCl or 2 murea. Even in 1 mNaCl, PCaP1
was stably associated with the membrane (data not
shown). PCaP1 was partially released from the mem-
brane by treatment with 0.1 mNa
2
CO
3
or 1% Tri-
ton X-100 (Fig. 3). In general, alkaline treatment with
Na
2
CO
3
removes peripheral membrane proteins, which
are associated with membrane intrinsic proteins, and a
mild detergent Triton X-100 is used to solubilize mem-
brane proteins, but not all membrane integral proteins.
Partial resistance to detergent and alkaline treatment
indicates that PCaP1 has properties similar to mem-
brane integral proteins.
Mode and sequence essential for membrane
association
The stable association of a protein without transmem-
brane domains with the plasma membrane led us to
determine the mode of interaction. The results shown
in Fig. 3 suggest that the interaction of PCaP1 with
the membrane does not occur electrostatically or by
association with transmembrane proteins. Indeed, we
failed to isolate a complex of PCaP1 with transmem-
brane protein(s). Therefore, we examined lipid modifi-
cation, especially N-myristoylation, as PCaP1 contains
a putative N-myristoylation consensus sequence,
Met-Gly-X-X-X-Ser-Lys, at the N-termini [4] (Fig. 1).
If the protein is N-myristoylated, Gly2 will be the
site of covalent modification. We prepared a PCaP1
mutant construct, whose Gly2 was replaced by Ala,
linked with green fluorescent protein (GFP) at the
C-terminus of PCaP1 (PCaP1
G2A
-GFP). We then
expressed the GFP fusion proteins in A. thaliana sus-
pension-cultured cells. We observed more than 25 cells
for each construct by confocal laser scanning micros-
copy. Green fluorescence of wild-type PCaP1 was
A
C
D
B
Fig. 2. Preparation of standard PCaP1 protein and immunochemical
quantification of PCaP1 in A. thaliana. (A) PCaP1 with (His)
6
tag
(His ⁄PCaP1) was expressed in Escherichia coli cells and purified
from the soluble fraction. Purified His ⁄PCaP1 was treated with
TAGZyme to remove the (His)
6
tag. Samples were subjected to
SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, solu-
ble fraction (10 lg) prepared from E. coli cells; lane 2, preparation
(1.5 lg) after nickel nitrilotriacetic acid Superflow column chroma-
tography; lane 3, TAGZyme system-treated fraction (1.5 lg); lane 4,
peak fraction (1.5 lg) after Sephacryl S-300 HR column chromatog-
raphy. Black and white arrowheads indicate the position of His ⁄P-
CaP1 (37 kDa) and PCaP1 (36 kDa), respectively. (B) Purified
PCaP1 (0, 5, 10, 15, 20, 30 and 40 ng) was subjected to SDS-
PAGE, followed by immunoblotting with anti-PCaP1 IgG. (C) Rela-
tive intensity of immunostained bands was plotted against the
amount of PCaP1 protein to prepare a calibration curve. (D) Crude
membrane (P100) and cytosol (S100) fractions were prepared from
shoots and roots of 2-week-old plants of A. thaliana by centrifuga-
tion at 100 000 g. The fractions (20 lg each) were subjected to
immunoblotting with anti-PCaP1 IgG (inset). The amount of PCaP1
protein on the basis of total protein in each fraction was calculated
using the standard curve.
A novel cation-binding myristoylated protein N. Nagasaki et al.
2270 FEBS Journal 275 (2008) 2267–2282 ª2008 The Authors Journal compilation ª2008 FEBS
clearly detected in the plasma membrane (Fig. 4A). In
contrast, the fluorescence of the PCaP1
G2A
mutant was
observed in the cytosol, but not in the plasma mem-
brane (Fig. 4B). This was not caused by the release of
the GFP moiety from the fusion protein by proteolytic
cleavage in the cells, because free GFP was always
localized to the cytosol and nucleus (data not shown).
To examine whether the N-terminal sequence included
a possible myristoylation signal, we prepared two addi-
tional GFP fusion proteins: one with the first
27-amino-acid sequence of PCaP1 (PCaP1
1)27
) and the
other with a modified N-terminal 27-residue sequence,
in which Gly2 was replaced by Ala (PCaP1
1)27 ⁄G2A
).
Green fluorescence of PCaP1
1)27
-GFP and
PCaP1
1)27 ⁄G2A
-GFP was detected in the plasma mem-
brane and cytosol, respectively (Fig. 4C,D). The results
indicate that the N-terminal part with 27 residues has
the ability to localize the protein to the plasma mem-
brane, and that Gly2 is essential for plasma membrane
localization.
In vitro myristoylation
To confirm the N-myristoylation of PCaP1, we carried
out an in vitro transcription ⁄translation assay in the
presence of [
3
H]myristic acid, using rabbit reticulocyte
lysate, which contained N-myristoyltransferase activity
[14]. Because N-myristoylation occurs cotranslational-
ly, the experiments were carried out in a cell-free tran-
scription ⁄translation system. CBL4 (also known as
A
B
C
D
Fig. 3. Tight association of PCaP1 with plasma membrane. (A) The
purified plasma membrane fraction was treated with 0.1 MNaCl,
2Murea, 0.1 MNa
2
CO
3
or 1% Triton X-100 for 20 min, and then
centrifuged as described in Experimental procedures. The PCaP1
contents in the supernatant (S) and pellet (P) were determined by
immunoblotting with anti-PCaP1 IgG. (B) The relative content of
PCaP1 in the supernatant and pellet was expressed as the percent-
age of the total amount of PCaP1. The data are the averages from
two independent experiments. (C) The purified plasma membranes
were treated with 0.1 MNa
2
CO
3
to release peripheral membrane
proteins (left) and HCl was added to neutralize the suspension
(right). (D) The suspensions were centrifuged at 100 000 g, and the
supernatant (S) and pellet (P) were subjected to immunoblotting
with anti-PCaP1 IgG.
µ
A
B
C
D
Fig. 4. Plasma membrane localization of PCaP1 variants. (A–D)
Expression of PCaP1-GFP fusion proteins in suspension-cultured
cells of A. thaliana. Constructs of PCaP1-GFP (A), PCaP1
G2A
-GFP
(B), PCaP1
1)27
-GFP (C) and PCaP1
1)27 ⁄G2A
-GFP (D) were transiently
expressed in the cells. Green fluorescence was viewed with a con-
focal laser scanning microscope (left panels). Nomarski images
were also recorded (right panels).
N. Nagasaki et al. A novel cation-binding myristoylated protein
FEBS Journal 275 (2008) 2267–2282 ª2008 The Authors Journal compilation ª2008 FEBS 2271
