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 ª 2008 The Authors Journal compilation ª 2008 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 PtdInsP species. 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 ª 2008 The Authors Journal compilation ª 2008 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 N ETPHOS 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 ª 2008 The Authors Journal compilation ª 2008 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 m NaCl or 2 m urea. Even in 1 m NaCl, PCaP1 was stably associated with the membrane (data not shown). PCaP1 was partially released from the mem- brane by treatment with 0.1 m Na 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 ª 2008 The Authors Journal compilation ª 2008 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 M NaCl, 2 M urea, 0.1 M Na 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 M Na 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 ª 2008 The Authors Journal compilation ª 2008 FEBS 2271 SOS3), a typical myristoylated protein, was synthesized as a 28 kDa protein, as detected by immunoblotting in this system, and was labelled with [ 3 H]myristic acid as shown by fluorography (Fig. 5). CBL4 is myristoylated at Gly2 in the N-terminal sequence (MGCSVSKKK) and functions as an EF-hand-type Ca 2+ -binding pro- tein [14]. When the Gly residue was replaced by Ala, the mutant CBL4 (CBL4 G2A ) did not incorporate [ 3 H]myristic acid. Both PCaP1 and its variant (PCaP1 G2A ) were translated and detected as 36 kDa proteins by immunoblotting. Radioactive [ 3 H]myristic acid was incorporated into PCaP1, but not into PCaP1 G2A (Fig. 5). These results indicate that PCaP1 is myristoylated at Gly2. Specific interaction of PCaP1 with PtdInsPs The N-terminal part of PCaP1 is rich in Lys and aro- matic (Tyr, Trp and Phe) residues (Fig. 1A). These characteristic sequences with clusters of basic ⁄ aromatic residues have been found in domains for interaction with PtdInsPs [8,15,16]. We determined the binding nature of PCaP1 using PIP Strips TM , which were spotted with a series of PtdInsPs (Fig. 6A). PCaP1 bound to phosphatidylino- sitol 3,4-bisphosphate [PtdIns(3,4)P 2 ], PtdIns(3,5)P 2 , PtdIns(4,5)P 2 and phosphatidylinositol 3,4,5-triphos- phate [PtdIns(3,4,5)P 3 ], and weakly with PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The protein did not associ- ate with lysophosphatidic acid, lysophosphatidylcho- line, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingosine 1-phosphate, phos- phatidic acid or phosphatidylserine. Next, we exam- ined the effect of calcium on these properties, because PCaP1 has been demonstrated to bind calcium [12]. The selectivity of binding was not changed by the addition of calcium to the reaction mixture at 4 lm (Fig. 6B). Potassium did not affect the intensity or selectivity, even at 10 mm, but magnesium weakened the affinity but not the selectivity. Further quantitative analysis was performed using a PIP Array TM (Fig. 6C). PCaP1 bound PtdInsPsina concentration-dependent manner (Fig. 6D). PCaP1 had a high affinity for PtdIns(3,5)P 2 and PtdIns(3,4,5)P 3 , and bound even at 3.1 pmol on the sheet. The affinity for PtdIns(3,4)P 2 and PtdIns(4,5)P 2 was relatively low. We examined the binding selectivity of PCaP1 using an array sheet containing PtdIns(3,5)P 2 , PtdIns(3,4)P 2 and PtdIns(4,5)P 2 (spot- ted by ourselves; data not shown), because there was a difference in the signal strength for the three PtdInsP 2 between the PIP Strips TM and PIP Array TM (Fig. 6A–C). This careful assay confirmed the high affinity of PCaP1 for PtdIns(3,5)P 2 , but not for PtdIns(3,4)P 2 or PtdIns(4,5)P 2 . The results indicated that PCaP1 has an ability to bind selectively to PtdIns(3,5)P 2 and PtdIns(3,4,5)P 3 amongst the various PtdInsPs. It should be noted that a (His) 6 tag did not affect the interaction of PCaP1 with PtdInsPs (data not shown). At present, we cannot deny the weak interaction with PtdIns(3,4)P 2 and PtdIns(4,5)P 2 in this in vitro assay system. Amino-terminal part of PCaP1 as the site of binding to PtdInsPs In general, the polybasic residue region is a good can- didate for binding to PtdInsPs. The N-terminal part is the most basic region, containing seven Lys residues. Thus, we carried out a competition assay of PtdInsP binding using a peptide that corresponds to the N-ter- minal part (positions 2–24) (Fig. 7E). The purified PCaP1 bound PtdInsP 2 and PtdIns(3,4,5)P 3 . The bind- ing intensity was decreased markedly in the presence of the PCaP1 2)24 peptide (Fig. 7A,B), suggesting that PCaP1 binds PtdInsPs at the N-terminal region. This possibility was confirmed by comparison of the Fig. 5. Incorporation of [ 3 H]myristic acid into PCaP1, but not into PCaP1 G2A , in a rabbit reticulocyte in vitro translation assay. In vitro transcription and translation of wild-type PCaP1 (WT) and PCaP1 G2A (G2A) were performed in the presence of [ 3 H]myristic acid. The translation products were subjected to SDS-PAGE, immunoblotting (right and middle panels) and fluorography (left panel). Constructs of CBL4 and its derivative with Gly2 replaced by Ala were exam- ined as positive and negative controls, respectively. The same incu- bation was performed without any template DNA (none). Arrowheads indicate the positions of PCaP1 (36 kDa) and CBL4 (27 kDa). Translation products were detected by immunoblotting with anti-PCaP1 (middle panel) and anti-(His) 6 (right panel) IgG. Molecular masses (kDa) of the standard proteins are shown on the right. A novel cation-binding myristoylated protein N. Nagasaki et al. 2272 FEBS Journal 275 (2008) 2267–2282 ª 2008 The Authors Journal compilation ª 2008 FEBS binding intensity to PtdInsPs of the wild-type and N-terminal truncated protein D2-25PCaP1 ⁄ His (Fig. 7E). The truncated form was slightly smaller than the wild- type PCaP1 and highly purified, as shown in Fig. 7F. This D2-25PCaP1 ⁄ His mutant protein gave no signal on either the PIP Strips TM or PI P A rray TM (Fig. 7C,D). Effect of calcium on the interaction of PCaP1 with the plasma membrane and PtdInsPs PCaP1 has been demonstrated to bind calcium by the 45 Ca-overlay assay [12]. Thus, we examined the effect of calcium on the association of PCaP1 with the plasma membrane. The purified plasma mem- branes did not release PCaP1, even in the presence of 10 mm CaCl 2 (Fig. 8A). Thus, calcium cannot be an effector or regulator for dissociation ⁄ association of PCaP1 from the membrane. As shown in the PIP Array TM test (Fig. 8B), calcium did not affect the interaction with PtdInsPs. The selectivity to PtdInsPs was unchanged and the interaction with PtdIns(3,5)P 2 and PtdIns(3,4,5)P 3 was retained up to 100 lm CaCl 2 . The affinity was decreased strongly in 1mm Ca 2+ and lost at 5 mm, concentrations way above the physiological concentration. Thus, binding to the plasma membrane and PtdInsPs may be stable in living cells at a physiological concentration of 0.1 lm [17,18]. Fig. 6. PCaP1 preferentially interacts with phosphatidylinositol di- and triphosphates. (A) Binding capacity of PCaP1 to PtdInsPs was tested with PIP Strips TM on which 15 kinds of lipid were immobilized (left). The strips were incubated in a solution contain- ing the purified recombinant PCaP1 (50 ngÆmL )1 ) (+PCaP1) or the buffer without the protein ()PCaP1) at 4 °C overnight (right). The strips were stained with anti- PCaP1 IgG, and the antigen PCaP1 bound to the strips was visualized. LPA, lysophos- phatidic acid; LPC, lysophosphocholine; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-monophosphate; PtdIns(4)P, phosphatidylinositol 4-mono- phosphate; PtdIns(5)P, phosphatidylinositol 5-monophosphate; PtdIns(3,4)P 2 , phosphati- dylinositol 3,4-bisphosphate; PtdIns(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P 3 , phos- phatidylinositol 3,4,5-triphosphate, PE, phosphatidylethanolamine; PC, phosphatidyl- choline; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (B) Incubation of PIP Strips TM with PCaP1 was carried out in the presence of KCl (b, c), MgCl 2 (d) or CaCl 2 (e). (C) Affinity of PCaP1 for individual lipids was determined using a PIP Array TM sheet (new version after 2004), on which lipids were immobi- lized at the indicated amount. (D) The signal intensities of a representative assay shown in (C) are expressed as a percentage of PtdIns(3,4,5)P 3 at 100 pmol. N. Nagasaki et al. A novel cation-binding myristoylated protein FEBS Journal 275 (2008) 2267–2282 ª 2008 The Authors Journal compilation ª 2008 FEBS 2273 Calcium-dependent interaction of PCaP1 with CaM There is no motif of enzymatic function in PCaP1. In order to understand the physiological role of a non- enzymatic protein, it is worth surveying the partner of the interaction protein. We examined the interaction of PCaP1 with CaM. When purified recombinant PCaP1 was incubated with CaM-agarose, PCaP1 bound to CaM-agarose, especially in the presence of Ca 2+ , and no PCaP1 was recovered in the unbound fraction or wash fraction (Fig. 9A, )CaM). This interaction was competitively inhibited by free CaM in the incubation medium (Fig. 9A, +CaM). The bound PCaP1 was released and eluted by an SDS solution (Fig. 9B). When free calcium was removed from the incubation medium by EGTA, no PCaP1 was bound to CaM- agarose (Fig. 9B). The results indicate that PCaP1 Fig. 7. An amino-terminal polybasic region is necessary for specific binding of PCaP1 to the phosphatidylinositol moiety. (A) The capacity of binding of PCaP1 to PtdInsP s (abbreviations as in Fig. 6) was tested with PIP Strips TM in the absence (left panel) or presence (right panel) of PCaP1 2)24 peptide. PCaP1 bound to the sheets was detected by immunoblotting with anti-PCaP1 IgG. (B) The signal intensities of a repre- sentative assay shown in (A) are expressed as a percentage of PtdIns(3,4,5)P 3 without the peptide. Wild-type (PCaP1) and N-terminal trun- cated PCaP1 (D 2-25PCaP1 ⁄ His) were assayed for PtdInsP binding capacity using PIP Strips TM (C) and PIP Array TM sheets (D). (E) Schematic diagram of PCaP1 2)24 peptide, PCaP1 and D2-25PCaP1 ⁄ His. Peptide sequence of PCaP1 2)24 is boxed. (F) SDS-PAGE profile of the purified D2-25PCaP1 ⁄ His. The D2-25PCaP1 ⁄ His protein was expressed in E. coli cells and purified from the soluble fraction. Samples were subjected to SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, soluble fraction (10 lg) prepared from the E. coli lysate; lane 2, preparation (1.5 lg) after nickel nitrilotriacetic acid Superflow column chromatography; lanes 3 and 5, peak fractions (1.5 lg) after HiTrap Phenyl HP col- umn chromatography (1.5 lg); lane 4, purified PCaP1 ⁄ His (1.5 lg). PCaP1 and D2-25PCaP1 ⁄ His were used in this assay. Black and white arrowheads indicate the position of PCaP1 ⁄ His and D2-25PCaP1 ⁄ His, respectively. A novel cation-binding myristoylated protein N. Nagasaki et al. 2274 FEBS Journal 275 (2008) 2267–2282 ª 2008 The Authors Journal compilation ª 2008 FEBS associates with CaM in a calcium-dependent manner. The membrane association of PCaP1 was not affected by CaM, even in the presence of Ca 2+ (Fig. 9C). How- ever, CaM suppressed the interaction of PCaP1 with PtdInsPs in the presence of Ca 2+ (Fig. 9D); the amount of PCaP1 bound to PtdInsPs was decreased to 25% as shown by the intensity of the PCaP1 signal in the PIP Array TM test. These results indicate that CaM binds with PCaP1 and affects the association of PCaP1 with PtdInsPs in a calcium-dependent manner. Discussion PCaP1 is a novel hydrophilic protein without a pre- dicted transmembrane domain in nature [12]. PCaP1 is a minor membrane component and accounts for 0.079% and 0.029% of the total membrane protein from shoots and roots, respectively, of A. thaliana seedlings (Fig. 2). The aim of this study was to clarify the mechanism of the specific tight association of PCaP1 with the plasma membrane in vivo and in vitro. Almost all PCaP1 was associated with the membrane and was not released by treatment with a high concen- tration of salt or urea (Fig. 3). Alkaline treatment with Na 2 CO 3 (pH 11.6) released PCaP1, but the released PCaP1 was recovered in the membrane by neutraliza- tion of the suspension with HCl (Fig. 3C,D), suggest- ing the involvement of basic residues, such as Lys (pK a for side-chain, 10.53), in the interaction with the membrane. The present study clearly reveals that N-myristoyla- tion anchors PCaP1 to the plasma membrane. First, when Gly2 of PCaP1 was replaced by Ala, the mutant PCaP1 was localized to the cytoplasm (Fig. 4). A Gly residue adjusted to the first Met is essential for N-myr- istoylation [4,19]. Second, the first 27 residues of the N-terminal sequence were sufficient for N-myristoyla- tion, as GFP linked with the 27-residue peptide was anchored to the membrane (Fig. 4). Third, [ 3 H]myristic acid was incorporated into PCaP1, but not into a PCaP1 G2A mutant (Fig. 5). Thus, we conclude that PCaP1 is myristoylated at Gly2 and that cotranslation- al myristoylation anchors the protein to the mem- brane. N-myristoylation is catalysed by two enzymes, namely methionine aminopeptidase (MAP) and myri- stoyl-CoA:protein N-myristoyltransferase (NMT). Three MAP isoforms, MAP1A, MAP2A and MAP2B, have been identified in A. thaliana as the cytoplasmic forms [20,21]. These MAPs catalyse the excision of the N-terminal Met residue from proteins. The subsequent myristoylation reaction is catalysed by N-myristoyl- transferase; for example, in A. thaliana, AtNMT1 has been demonstrated to modify several known N-myri- stoylated proteins in vitro [4]. A comprehensive study of the substrate specificity of AtNMT1 has revealed that the positive charge on residue 7 of the sub- strate proteins is particularly important. The seventh N-terminal residue of PCaP1 is Lys (Fig. 1). Thus, PCaP1 may be cotranslationally N-myristoylated by a A B Fig. 8. Association of PCaP1 with the plasma membrane and PtdInsPs (abbreviations as in Fig. 6) in the presence of calcium. (A) The puri- fied plasma membranes were incubated with CaCl 2 at the indicated concentrations at room temperature for 20 min, and then centrifuged at 100 000 g for 15 min. Both the supernatant (S) and precipitate (P) fractions (20 lg of protein in each lane) were subjected to immunoblotting with anti-PCaP1 IgG. (B) PIP Array TM sheets were incubated with the purified recombinant PCaP1 (50 ngÆmL )1 ) in the absence (a) or pres- ence (b–f) of CaCl 2 . The bound PCaP1 was detected with anti-PCaP1 IgG. N. Nagasaki et al. A novel cation-binding myristoylated protein FEBS Journal 275 (2008) 2267–2282 ª 2008 The Authors Journal compilation ª 2008 FEBS 2275 cytoplasmic MAP and AtNMT1, and subsequently anchored to the cytoplasmic face of the plasma mem- brane. Furthermore, it is clear from the present study that the N-terminal 27-residue sequence is necessary and sufficient for the N-myristoylation of PCaP1. PCaP1 is a novel protein with unique structural fea- tures, namely an abundance of Glu and Lys residues and a lack of common functional motifs. A previous study has suggested the constitutive expression and significant stimulation of gene expression by a patho- logical elicitor (flagellin peptide) and copper [12]. In general, N-myristoylation provides the primary mem- brane-targeting signal for several plant protein kinases, such as zucchini CpCDPK1, A. thaliana AtCPK2 and tomato LeCPK1 [6,22–25]. A plant Rab GTPase, Ara6, which plays a critical role in endosomal homo- typic fusion, also requires N-myristoylation for its endosomal localization [26]. In our preliminary experi- ments, T-DNA insertion mutant lines of PCaP1 showed decreased tolerance to pathological infection and heavy metal ion stresses. PCaP1 may be involved in the intracellular response to some physiological stresses. The biochemical role of PCaP1 remains to be examined, with a consideration of the phenotypic properties of the knockout mutant plants. The elucidation of the specific interaction with PtdInsPs provides essential information for an under- standing of the physiological role of PCaP1 in plants. A large number of proteins associate with PtdInsPsin membranes with high or low specificity, and express their own activities, such as intracellular signalling and organization [8,15,16]. In eukaryotic cells, PtdInsPs constitute a minor fraction of total membrane lipid, but play many important roles [27,28]. We demon- AB C D Fig. 9. Interaction of PCaP1 with CaM and its effect on binding with PtdInsPs (abbreviations as in Fig. 6). (A) Purified PCaP1 was mixed with CaM-agarose in the presence (lanes 5–8) or absence (lanes 1–4) of CaM (top panel). The same experiments were performed in the presence of 0.5 m M Ca 2+ (middle) or 1 mM EGTA (bottom). After centrifugation, the supernatant fractions (Ub, unbound fraction; lanes 1 and 5) were collected. The CaM-agarose beads were washed three times with the same buffer. The supernatants obtained (W1, W2, W3; lanes 2–4 and 6–8) and the unbound fraction were subjected to SDS-PAGE and protein staining. (B) PCaP1 was incubated with CaM-agarose in the presence of 0.5 m M Ca 2+ (lanes 1 and 2) or 1 mM EGTA (lanes 3 and 4). CaM was added to the mixture (lanes 2 and 4). Proteins bound to CaM-agarose were released with an SDS solution and subjected to SDS-PAGE. Lane 5, recombinant PCaP1 (0.0175 lg). (C) The purified plasma membranes were incubated with or without 0.167 mgÆmL )1 CaM, 0.1 m M CaCl 2 and 1 m M EGTA at room temperature for 20 min. After centrifugation at 100 000 g for 15 min, aliquots (20 lg) of the supernatant (S) and precipitated (P) fractions were subjected to immunoblotting with anti-PCaP1 IgG. (D) PIP Array TM sheets were incubated with purified recombinant PCaP1 (50 ngÆmL )1 ) in the presence of 0.167 mgÆmL )1 CaM, 0.1 mM CaCl 2 and ⁄ or 1 mM EGTA. Bound PCaP1 was detected by immunoblotting. A novel cation-binding myristoylated protein N. Nagasaki et al. 2276 FEBS Journal 275 (2008) 2267–2282 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... observation that Ca2+ ⁄ CaM suppresses the PCaP1–PtdInsP interaction can be explained In conclusion, we have demonstrated that PCaP1 tightly binds to the plasma membrane via N-myristoylation at Gly2 and specifically interacts with PtdIns(3,5)P2 and PtdIns(3,4,5)P3 in the membrane N-myristoylation anchors PCaP1, and the interaction with PtdInsPs may contribute to the stability of the attachment of PCaP1 to. .. study has revealed that CaM interacts with PCaP1 in a calcium-dependent manner (Fig 9) CaM, an acidic protein with four high-affinity Ca2+binding sites, is well known as the protein mediator of many Ca2+-stimulated enzymes, such as phosphoinositide 3-kinase and plasma membrane Ca2+-ATPase The presence of CaM and Ca2+ suppressed the interaction of PCaP1 with PtdInsPs The binding of PCaP1 to the plasma membrane. .. membrane was not affected by CaM, even in the presence of Ca2+ Therefore, CaM and Ca2+ may regulate the unidentified function of PCaP1 localized in the plasma membrane It has been reported that a brain-specific protein CAP-23 ⁄ NAP22 is myristoylated and interacts with Ca2+ ⁄ CaM at the myristoylated N-terminal domain [40] If Ca2+ ⁄ CaM binds to the N-terminal site of PCaP1 competitively with PtdInsPs, the. .. glycerol and 1 mm dithiothreitol, and used as a crude membrane fraction Plasma membranes were isolated from the crude membranes with an aqueous two-phase partitioning system [42,44] Crude membranes were prepared from taproots of R sativus and B rapa, petioles of B rapa var glabra Regel and shoots of B oleracea var italica by the same methods, and used for immunoblotting Preparation of recombinant PCaP1... Histochem Cytochem 50, 697–708 42 Kobae Y, Sekino T, Yoshioka H, Nakagawa T, Martinoia E & Maeshima M (2006) Loss of AtPDR8, a plasma membrane ABC transporter of Arabidopsis thaliana, causes hypersensitive cell death upon pathogen infection Plant Cell Physiol 47, 309–318 43 Ishikawa F, Suga S, Uemura T, Sato MH & Maeshima M (2005) Novel type aquaporin SIPs are mainly localized to the ER membrane and. .. to the membrane It has been shown that the attachment to the membrane is stable under physiological conditions (Fig 8), and that Ca2+ ⁄ CaM regulates the association of PCaP1 with PtdInsPs (Fig 9) Many proteins are N-myristoylated or interact with PtdInsPs in various organisms PCaP1 is a highly unique protein, because the N-terminal domain is required for both N-myristoylation and the specific interaction... The vector, pET ⁄ PCaP1, was then directly amplified by PCR with a pair of primers (forward, 5¢-CACCACCACCACCAGATGGGTTACTGGAATTCCA AG-3¢; reverse, 5¢-GTGGTGTTTCATATGTATATCTCCT TCTTAAAGTTAAAC-3¢; italic type shows the His-tag adaptor sites) After confirmation of the nucleotide sequences of the pET ⁄ His ⁄ PCaP1 obtained, the expression vector was introduced into E coli BL21(DE3) (Novagen) Transformants... MS-sucrose) A thaliana suspension-cultured cells (also known as ‘Deep’ cells) were a kind gift from Masaaki Umeda (University of Tokyo, Japan) The cells were cultured in MS medium at 22 °C in the dark Other plants [Raphanus sativus (radish), Brassica rapa (turnip), Brassica rapa L var glabra Regel (Chinese cabbage) and Brassica oleracea var italica (broccoli)] were purchased from a market Purification of recombinant... Plasma membrane targeting and biochemical characterization Plant Physiol 129, 156– 168 25 Martin ML & Busconi L (2000) Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation Plant J 24, 429–435 26 Ueda T, Yamaguchi M, Uchimiya H & Nakano A (2001) Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis. .. from the membrane fraction Both fractions were subjected to immunoblotting with polyclonal antibody to PCaP1, which was prepared previously [12] Membrane association assay The purified plasma membranes were incubated with CaCl2 at room temperature for 20 min, and then centrifuged at 100 000 g for 15 min to determine the effect of calcium on the association of PCaP1 with the plasma membrane In some cases, . A hydrophilic cation-binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N-myristoylation and interacts with calmodulin. 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