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• W2040642807 abstract "PV72, a type I membrane protein with three epidermal-growth factor (EGF)-like motifs, was found to be localized on the membranes of the precursor-accumulating (PAC) vesicles that accumulated precursors of various seed storage proteins. To clarify the function of PV72 as a sorting receptor, we expressed four modified PV72s and analyzed their ability to bind the internal propeptide (the 2S-I peptide) of pro2S albumin by affinity chromatography and surface plasmon resonance. The recombinant PV72 specifically bound to the 2S-I peptide with a K D value of 0.2 μm, which was low enough for it to function as a receptor. The EGF-like motifs modulated the Ca2+-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The binding of Ca2+ stabilizes the receptor-ligand complex even at pH 4.0. The association and dissociation of PV72 with the ligand is modulated by the Ca2+ concentration (EC50 value = 40 μm) rather than the environmental pH. Overall results suggest that Ca2+ regulates the vacuolar sorting mechanism in higher plants. PV72, a type I membrane protein with three epidermal-growth factor (EGF)-like motifs, was found to be localized on the membranes of the precursor-accumulating (PAC) vesicles that accumulated precursors of various seed storage proteins. To clarify the function of PV72 as a sorting receptor, we expressed four modified PV72s and analyzed their ability to bind the internal propeptide (the 2S-I peptide) of pro2S albumin by affinity chromatography and surface plasmon resonance. The recombinant PV72 specifically bound to the 2S-I peptide with a K D value of 0.2 μm, which was low enough for it to function as a receptor. The EGF-like motifs modulated the Ca2+-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The binding of Ca2+ stabilizes the receptor-ligand complex even at pH 4.0. The association and dissociation of PV72 with the ligand is modulated by the Ca2+ concentration (EC50 value = 40 μm) rather than the environmental pH. Overall results suggest that Ca2+ regulates the vacuolar sorting mechanism in higher plants. epidermal growth factor an internal propeptide of pumpkin 2S albumin clathrin-coated vesicles precursor-accumulating vesicles 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 4-morpholineethanesulfonic acid Most proteins that are synthesized on rough endoplasmic reticulum are delivered to various cellular destinations, including vacuoles and lysosomes. Such sorting involves recognition of targeting signals of proteins by receptors. In mammalian systems, mannose 6-phosphate residues in glycosyl side chains of glycoproteins are known to function as a targeting signal to the lysosomes, and mannose 6-phosphate receptors have been identified as a lysosome sorting receptor (1Dahms N.M. Lobel P. Kornfeld S. J. Biol. Chem. 1989; 264: 12115-12118Abstract Full Text PDF PubMed Google Scholar). In yeast systems, a short stretch sequence of amino acids, QRPL, found in the carboxypeptidase Y, is known to function as a targeting signal to the vacuoles, and Vps10p has been identified as a vacuolar sorting receptor for vacuolar hydrolases (2Marcusson E.G. Horazdovsky B.F. Cereghino J.L. Gharakhanian E. Emr S.D. Cell. 1994; 77: 579-586Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Higher plants have two types of vacuoles: one type, protein storage vacuoles, develop mainly in storage organs, such as seeds, and the other type, lytic vacuoles, which contain various lytic enzymes, develop in the vegetative organs. Both types of vacuoles, however, are found in the same cells of barley roots (3Paris N. Stanley C.M. Jones R.L. Rogers J.C. Cell. 1996; 85: 563-572Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar) and of maturing pea seeds (4Hoh B. Hinz G. Jeong B.-K. Robinson D.G. J. Cell Sci. 1995; 108: 299-310PubMed Google Scholar). In these cells, vacuolar proteins synthesized on the rough endoplasmic reticulum are sorted and delivered to their respective vacuoles. Thus, different targeting machinery for each type of vacuole must be involved in protein transport in these cells. For the lytic vacuoles, BP-80 was the first putative vacuolar sorting receptor isolated from pea (5Kirsch T. Paris N. Butler J.M. Beevers L. Rogers J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3403-3407Crossref PubMed Scopus (191) Google Scholar). It binds in vitro to the vacuolar-targeting determinants (6Kirsch T. Saalbach G. Raikhel N.V. Beevers L. Plant Physiol. 1996; 111: 469-474Crossref PubMed Scopus (95) Google Scholar, 7Cao X. Rogers S.W. Butler J. Beevers L. Rogers J.C. Plant Cell. 2000; 12: 493-506Crossref PubMed Scopus (84) Google Scholar). Recently, Humair et al. (8Humair D. Felipe D.H. Neuhaus J.M. Paris N. Plant Cell. 2001; 13: 781-792Crossref PubMed Scopus (62) Google Scholar) demonstrated the in vivo binding of BP-80 to the propeptide sequence of barley aleurain in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. An Arabidopsis homolog, AtELP, was also found to interact with the propeptide of an aleurain homolog, AtALEU (9Ahmed S.U. Rojo E. Kovalentina V. Venkataraman S. Dombrowski J.E. Matsuoka K. Raikhel N.V. J. Cell Biol. 2000; 149: 1335-1344Crossref PubMed Scopus (171) Google Scholar). BP-80 (10Paris N. Rogers A.W. Jiang L. Kirsch T. Beevers L. Phillips T.E. Rogers J.C. Plant Physiol. 1997; 115: 29-39Crossref PubMed Scopus (164) Google Scholar) and AtELP (11Ahmed S.U. Bar-Peled M. Raikhel N.V. Plant Physiol. 1997; 114: 325-336Crossref PubMed Scopus (128) Google Scholar) are a type I integral membrane protein with epidermal growth factor (EGF)1-like motifs. They have been shown to be rich in clathrin-coated vesicles (CCVs) and prevacuolar compartments (12Hinz G. Hilmer S. Baumer M. Hohl I. Plant Cell. 1999; 11: 1509-1524Crossref PubMed Scopus (114) Google Scholar, 13Sanderfoot A.A. Ahmed S.U. Marty-Mazars D. Rapoport I. Kirchhausen T. Marty F. Raikhel N.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9920-9925Crossref PubMed Scopus (150) Google Scholar). This implied that the cysteine proteinases might be delivered from the Golgi complex to lytic vacuoles via the CCVs in a receptor-dependent manner (9Ahmed S.U. Rojo E. Kovalentina V. Venkataraman S. Dombrowski J.E. Matsuoka K. Raikhel N.V. J. Cell Biol. 2000; 149: 1335-1344Crossref PubMed Scopus (171) Google Scholar, 14Jiang L. Rogers J.C. J. Cell Biol. 1998; 143: 1183-1199Crossref PubMed Scopus (184) Google Scholar). In contrast, the CCVs isolated from maturing pea seeds were reported to contain no storage proteins (12Hinz G. Hilmer S. Baumer M. Hohl I. Plant Cell. 1999; 11: 1509-1524Crossref PubMed Scopus (114) Google Scholar). Therefore, the transport machinery for storage proteins should be different from that of the lytic enzymes. However, the molecular mechanism responsible for the transport of storage proteins are scarcely elucidated. In maturing seeds of plants, seed storage protein precursors are synthesized on the rough endoplasmic reticulum and then transported to protein storage vacuoles, where the precursor proteins are converted into the respective mature forms by the action of vacuolar processing enzyme (15Hara-Nishimura I. Nishimura M. Plant Physiol. 1987; 85: 440-445Crossref PubMed Google Scholar, 16Hara-Nishimura I. Inoue K. Nishimura M. FEBS Lett. 1991; 294: 89-93Crossref PubMed Scopus (210) Google Scholar, 17Hara-Nishimura I. Takeuchi Y. Nishimura M. Plant Cell. 1993; 5: 1651-1659Crossref PubMed Scopus (135) Google Scholar, 18Hara-Nishimura I. Shimada T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1995; 145: 632-640Crossref Scopus (100) Google Scholar, 19Hara-Nishimura I. Kinoshita T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1998; 152: 668-674Crossref Scopus (63) Google Scholar, 20Kinoshita T. Yamada K. Hiraiwa N. Nishimura M. Hara-Nishimura I. Plant J. 1999; 19: 43-53Crossref PubMed Scopus (180) Google Scholar). Multiple transport pathways have been shown for storage proteins. Hohl et al. (21Hohl I. Robinson D.G. Chrispeels M.J. Hinz G. J. Cell Sci. 1996; 109: 2539-2550PubMed Google Scholar) and Hinz et al. (12Hinz G. Hilmer S. Baumer M. Hohl I. Plant Cell. 1999; 11: 1509-1524Crossref PubMed Scopus (114) Google Scholar) demonstrated immunocytochemically that dense vesicles with a diameter of about 100 nm associated with Golgi complex contain storage proteins in maturing pea cotyledons. We found the other unique vesicles responsible for delivery of precursors of seed storage proteins and a membrane protein into the vacuoles (22Hara-Nishimura I. Nishimura M. Akazawa T. Plant Physiol. 1985; 77: 747-752Crossref PubMed Google Scholar, 23Fukasawa T. Hara-Nishimura I. Nishimura M. Plant Cell Physiol. 1988; 29: 339-345Google Scholar, 24Hara-Nishimura I. Takeuchi Y. Inoue K. Nishimura M. Plant J. 1993; 4: 793-800Crossref PubMed Scopus (120) Google Scholar) and designated them PAC (precursor-accumulating) vesicles (25Hara-Nishimura I. Shimada T. Hatano K. Takeuchi Y. Nishimura M. Plant Cell. 1998; 10: 825-836Crossref PubMed Scopus (246) Google Scholar). The PAC vesicles are derived from the endoplasmic reticulum and mediate a transport for storage proteins directly to protein storage vacuoles. We have found an integral membrane protein, PV72, with EGF-like motifs in the PAC vesicle fraction prepared from maturing pumpkin seeds (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar). We have also shown that PV72 exhibits an affinity for peptides derived from pumpkin 2S albumin (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar). The question is how the association and dissociation of a receptor and the respective ligand is regulated. In mammalian systems, the affinity of mannose 6-phosphate receptor for their glycoprotein ligands was reported to be reduced by the acidic pH of the lysosomes (1Dahms N.M. Lobel P. Kornfeld S. J. Biol. Chem. 1989; 264: 12115-12118Abstract Full Text PDF PubMed Google Scholar). From the analogy to the system, the binding of BP-80 and AtELP to the ligand was described to be regulated by the pH (5Kirsch T. Paris N. Butler J.M. Beevers L. Rogers J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3403-3407Crossref PubMed Scopus (191) Google Scholar, 9Ahmed S.U. Rojo E. Kovalentina V. Venkataraman S. Dombrowski J.E. Matsuoka K. Raikhel N.V. J. Cell Biol. 2000; 149: 1335-1344Crossref PubMed Scopus (171) Google Scholar). In this study, however, we found that PV72 still has an ability to bind to the ligand at pH 4.0 in the presence of Ca2+ and that the EGF-like motifs modulate a Ca2+-dependent conformational change of PV72. We describe here a unique mechanism by which Ca2+ acts as a modulator for the association and dissociation of PV72 with its ligand. Pumpkin (Cucurbita sp cv Kurokawa Amakuri Nankin) seeds were purchased from Aisan Shubyo Seed Co. (Nagoya, Japan). The seeds were planted in the field. The cotyledons of maturing seeds, freshly harvested 22–28 days after anthesis, were used for the experiments. PAC vesicles were isolated from pumpkin cotyledons at the middle stage of seed maturation as described previously (25Hara-Nishimura I. Shimada T. Hatano K. Takeuchi Y. Nishimura M. Plant Cell. 1998; 10: 825-836Crossref PubMed Scopus (246) Google Scholar). The cotyledons were homogenized in buffer A (20 mm sodium pyrophosphate, pH 7.5, 1 mm EDTA and 0.3 m mannitol) with an ice-chilled mortar and pestle and filtered through three layers of cheesecloth. The filtrate was centrifuged at 3,000 g for 15 min and the supernatant was centrifuged again at 8,000 g for 20 min at 4 °C. The pellet was suspended in buffer B (10 mm HEPES-KOH, pH 7.2, 1 mm EDTA and 0.3m mannitol) and layered on 28% Percoll (Amersham Biosciences, Tokyo, Japan) solution. After centrifugation at 40,000 × g for 35 min, the vesicle fraction was pooled and washed once in buffer B. PAC vesicles were collected by the centrifugation at 10,000 × g for 20 min and resuspended in buffer B. The isolated vesicles were subjected to immunoblot analysis. Maturing pumpkin seeds were vacuum-infiltrated for 1 h with a fixative that consisted of 4% paraformaldehyde, 1% glutaraldehyde, and 0.06m sucrose in 0.05 m cacodylate buffer, pH 7.4. The tissues were then cut into slices of less than 1 mm in thickness with a razor blade and treated for another 2 h with freshly prepared fixative. Immunoblot analysis was performed essentially as described previously (27Mitsuhashi N. Shimada T. Mano S. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 2000; 41: 993-1001Crossref PubMed Scopus (121) Google Scholar), except that in the present study we used specific antibodies against PV72 (diluted 5,000-fold) (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar) and horseradish peroxidase-conjugated donkey antibodies against rabbit IgG (diluted 5,000-fold; Amersham Biosciences, Inc.). PV72 was immunologically detected with an enhanced chemiluminescence kit (an ECL system, Amersham Biosciences, Inc.). Four modified PV72s were expressed in insect cells of Spodoptera frugiperda (Sf21) with a baculovirus expression system (Invitrogen, San Diego, CA). The system includes a transfer vector pBlueBac 4.5 and an expression vector Bac-N-Blue DNA composed of engineered baculoviral Autographa california multiple polyhedrosis virus. The KpnI-SacI fragment of pPV72 was produced from the amplified DNA with PV72 cDNA and a unique primer of 5′-ATT TGT TTA ACT GAA GAC GTG CAC CAC CAC CAC CAC CAC GAT GAG CTT TGA GGT ACC GAA TTC-3′ and was ligated with theKpnI-SacI-digested pBlueBac 4.5 to produce the pBlueBac-rPV72. The pBlueBac-rPV72 encoded a fusion protein composed of both the signal sequence and the lumen domain of PV72 followed by a polyhistidine tag and an HDEL sequence. Constructs for three other modified PV72s were produced by the same procedure described above, except for using each primer: 5′-GAT GGA GTC CAC ACG TGT GAA CAC CAC CAC CAC CAC CAC GAT GAG CTT TGA GGT ACC GAA TTC-3′ for PV72Δ3, 5′-YAC ACT CAT TGT GAA GCT CAC CAC CAC CAC CAC CAC GAT GAG CTT TGA GGT ACC GAA TTC-3′ for PV72Δ2,3, and 5′-ATT TGT TTA ACT GAA CAC CAC CAC CAC CAC CAC GAT GAG CTT TGA GGT ACC GAA TTC-3′ for PV72Δ1,2,3. We cotransfected Sf21 cells with Bac-N-Blue DNA and each of the produced plasmids to generate recombinant baculoviruses. The viruses were purified from the supernatant of the transfected cells by a plaque assay to generate a high titer recombinant viral stock (28De Hayashi H. Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). For large scale expression of these modified PV72s, the optimal expression time was determined by monitoring the cellular extract using SDS-PAGE and an immunoblot with anti-PV72 antibodies. Three days after infection, the cells were collected by centrifugation at 500 g for 10 min, washed with PBS and gently suspended in 20 mmHEPES-NaOH, pH 7.0, 150 mm NaCl, 1% CHAPS, and 1 mm CaCl2. The cells were lysed by three bursts of sonication for 1 min at 10-min intervals on ice and were centrifuged at 750,000 × g for 30 min. The supernatant was loaded on a Hi-Trap chelating column (Amersham Biosciences, Inc.) and was eluted with a gradient of 20–1,000 mm imidazol in the above buffer. The modified PV72 fractions were dialyzed against the HEPES buffer (20 mm HEPES-NaOH, pH 7.0, 150 mmNaCl, and 0.4% CHAPS) plus 1 mm CaCl2 were concentrated using Centricon 30 (Amicon Inc., Beverly, MA) and then were loaded on a Superdex-200 column (Amersham Biosciences, Inc.) equilibrated with the HEPES buffer plus 1 mmCaCl2. The purified modified PV72s were concentrated by Centricon 30 and subjected to a protein assay (Nippon Bio-Rad Laboratories, Tokyo) and a binding assay as described below. Five peptides (SRDVLQMRGIENPWRREG (2S-I), SRDVLQMRGIENPWGGGG (2S-I/3G), SRDVLQMRGIENGWRREG (2S-I/P75G), SRDVLQMRGIGNPWRREG (2S-I/E73G), SRDVLQMRGIENPWRRGG (2S-I/E79G)) were chemically synthesized with a peptide synthesizer (model 431A; Applied Biosystems Inc., Tokyo, Japan) and were used for ligands of affinity columns. Each peptide (10 mg) was immobilized to N-hydroxysuccinimide-activated Sepharose HP (Amersham Biosciences, Inc.) to prepare affinity columns. The modified PV72s were applied to each column equilibrated with the HEPES buffer plus 1 mm CaCl2 and then eluted with the HEPES buffer plus 2.5 mm EDTA (20 mmHEPES-NaOH, pH 7.0, 150 mm NaCl, 0.4% CHAPS, and 2.5 mm EDTA) with an automated chromatography system (ÄKTA, Amersham Biosciences, Inc.). Each fraction was subjected to SDS-PAGE and subsequently to an immunoblot analysis with anti-PV72 antibodies. We immobilized either 2S-I peptide or 2S-I/3G peptide on a sensor chip for a BIACORE system (BIACORE, Tokyo, Japan) in 10 mmHEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA, and 0.005% P-20 (HBS, BIACORE). Carboxymethylated dextran on a sensor chip (CM5) was activated with the mixture (70 μl) of 0.05m N-hydroxysuccinimide and 0.05 m N-ethyl-N-(3-diethylaminopropyl)carbodiimide and then coupled with either 2S-I peptide or 2S-I/3G peptide at 25 °C and a flow rate of 5 μl/min for the solutions used on a BIACORE system. A control flow cell was prepared with no peptide. The amount of the coupled peptide on the sensor chip was found to be 1200–1500 resonance units. The modified PV72s were injected onto the sensor chip for 300 s, and then the HEPES buffer plus 1 mm CaCl2 was eluted for 200–300 s at 25 °C and a flow rate of 30 μl/min. The sensor chip surface was regenerated with 30 μl of 20 mmHCl to remove residual PV72 from the immobilized peptides. Equal volumes of each protein dilution were also injected over a control flow cell to serve as blank sensorgrams for subtraction of bulk refractive index background and nonspecific binding of analyte. The sensorgrams shown in this study are made by subtracting the sensorgram made with the control flow cell. Kinetic analysis was performed according to the manufacturer's protocol. The association, dissociation, and regeneration phases were followed in real time as the changes in the relative diffraction. The association phase (0–180 s) was analyzed by nonlinear least squares curve fitting to yield the association rate constants (k a) as mean values. The dissociation phase (180–300 s) was also analyzed by nonlinear least squares curve fitting to yield the dissociation rate constants (k d). To avoid mass transport, we worked at a low immobilization level, a high flow rate (30 μl/min), and using suitable concentrations of analyte. Kinetic constants (the association rate constant (k a), the dissociation rate constant (k d), and the dissociation constant (K D = k d/k a)) were calculated from the sensorgrams using BIA evaluation software version 2.1 (BIACORE). These kinetic parameters were determined from three independent experiments. Ca2+-dependent binding was analyzed by two methods: surface plasmon resonance analysis and affinity chromatography. Both rPV72 and rPV72Δ1,2,3 were dialyzed against the HEPES buffer plus 2.5 mm EGTA, followed by dialysis against the HEPES buffer to remove Ca2+ and EGTA. The dialyzed PV72s were injected onto the 2S-I-immobilized sensor chip equilibrated with the HEPES buffer plus 0, 0.02, 0.05 0.1, and 1 mm CaCl2 or 1 mm MgCl2to obtain each sensorgram. Alternatively rPV72 was applied to the 2S-I affinity column with the HEPES buffer plus 1 mm CaCl2 and then washed with the HEPES buffer containing decreasing concentration of CaCl2 (500, 100, 50, 20, 0 μm). Finally, the column was eluted with the HEPES buffer plus 1 mm EGTA. To compare the Ca2+ sensitivity of rPV72 with that of rPV72Δ1,2,3, both modified proteins were applied to the 2S-I column with either the HEPES buffer plus 1 mm CaCl2 or the MES buffer (20 mm MES-NaOH, pH 5.5, 150 mmNaCl, 0.4% CHAPS) plus 1 mm CaCl2. We used the HEPES buffer plus 50 μm CaCl2 or the MES buffer plus 50 μm CaCl2 as the washing solution for the columns and the HEPES buffer plus 2.5 mmEGTA or the MES buffer plus 2.5 mm EGTA as the elution buffers. Each fraction was subjected to SDS-PAGE and subsequently to an immunoblot analysis. To investigate the pH effect on the interaction of modified PV72s with either 2S-I peptide, 2S-I/E73G peptide, 2S-I/P75G peptide, and 2S-I/E79G peptide, the proteins were subjected to each affinity column equilibrated with the HEPES buffer plus 1 mm CaCl2 or the sodium acetate buffer (20 mm sodium acetate, pH 4.0, 150 mm NaCl, 0.4% CHAPS) plus 1 mmCaCl2. The column was washed with the respective buffer and eluted with the respective buffer plus 2.5 mm EGTA. Each fraction was subjected to SDS-PAGE and subsequently to an immunoblot analysis. Fluorescence emission spectra were recorded from 300 to 400 nm by a fluorescence spectrophotometer (Hitachi, F-4500, Tokyo, Japan) with an excitation wavelength at 280 nm in mixtures containing 1 μg/ml modified PV72s, in the HEPES buffer plus 1 mm CaCl2 or the HEPES buffer plus 1 mm EDTA, as described previously (29Schmid F.X. Creighton T.E. Protein Structure: A Practical Approach. Oxford University Press, Oxford1989: 251-285Google Scholar). Previously, we found unique vesicles, PAC vesicles, which mediate the transport of the storage protein precursors to protein storage vacuoles in maturing pumpkin seeds (25Hara-Nishimura I. Shimada T. Hatano K. Takeuchi Y. Nishimura M. Plant Cell. 1998; 10: 825-836Crossref PubMed Scopus (246) Google Scholar). Electron microscopy of the maturing seeds revealed numerous electron-dense PAC vesicles within the cells, as indicated byarrowheads in Fig. 1. Isolation of the PAC vesicles showed that they accumulated proprotein precursors of seed storage proteins, but not their mature forms at all (Fig. 2, lane 1). The precursors included pro2S albumin, proglobulin, and PV100, which is a single precursor of multifunctional proteins including trypsin inhibitors, cytotoxic peptides, and 7S globulin (30Yamada K. Shimada T. Kondo M. Nishimura M. Hara-Nishimura I. J. Biol. Chem. 1999; 274: 2563-2570Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). PV72 was detectable as a single band with a molecular mass of 72 kDa on an immunoblot of the PAC vesicles with anti-PV72 antibodies (Fig. 2,lane 2). The PV72 content was enough high to be visible on the SDS gel with Coomassie Blue staining (Fig. 2, lane 1). The result indicates that the pure PAC vesicles contain PV72 as the fourth abundant protein of the vesiclesFigure 2Localization of PV72 in PAC vesicles that accumulate a proprotein precursor of 2S albumin, a seed storage protein. Isolated PAC vesicles were subjected to SDS-PAGE and subsequent staining with Coomassie Blue (lane 1) or immunoblot analysis with anti-PV72 antibodies (lane 2).p2S, pro2S albumin; pG, proglobulin;PV100, a precursor of a proteinase inhibitor, cytotoxic proteins, and 7S globulin (30Yamada K. Shimada T. Kondo M. Nishimura M. Hara-Nishimura I. J. Biol. Chem. 1999; 274: 2563-2570Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Lane M contains molecular mass markers. The molecular mass of each marker protein is given on theleft in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To clarify the ligand-binding mechanism of PV72, we expressed modified rPV72s with a His tag in insect Sf21 cells employing a baculovirus expression system. PV72 is a type I integral membrane protein with EGF-like motifs (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar). Fig. 3 A shows each construct of the modified proteins; rPV72 corresponds to the lumenal domain of PV72, rPV72Δ3 corresponds to the lumenal domain without the third EGF-like motif, rPV72Δ2,3 corresponds to the lumenal domain without the second and third EGF-like motifs, and rPV72Δ1,2,3 corresponds to the lumenal domain with no EGF-like motifs. These expressed proteins were purified with a chelating column and a gel filtration column. Each final preparation was highly pure as judged from SDS-PAGE with Coomassie Blue staining (Fig. 3 B). All of their N-terminal amino acid sequences were determined to be RFVVEKNSLK, which corresponds to the N-terminal sequence of authentic pumpkin PV72 as reported by Shimada et al. (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar). The results indicate that a signal peptide of the expressed proteins is correctly processed on the rough endoplasmic reticulum. To investigate the binding ability of rPV72 to the ligand peptides, we performed a binding assay on the affinity column (2S-I column) that was conjugated with the 2S-I peptide, the internal propeptide of pumpkin 2S albumin. As shown in Fig. 4 A(upper), rPV72 bound to the 2S-I column and then eluted with EDTA (discussed below). Previously we reported that the isolated PV72 from the maturing seeds of pumpkin binds to the 2S-I peptide but not to the mutant peptide 2S-I/3G with GGG instead of RRE of the internal propeptide. To clarify the specificity of the binding of rPV72, we used another affinity column (2S-I/3G column) that was conjugated with the 2S-I/3G peptide. As shown in Fig. 4 A (lower), rPV72 did not bind to the 2S-I/3G column. The results indicate that the characteristics of rPV72 with respect to ligand binding were the same as those of the authentic PV72 has. To identify the ligand-binding region of PV72, we performed a surface plasmon resonance analysis for four modified PV72s with either the 2S-I sensor chip or 2S-I/3G sensor chip. Each modified PV72 of the same concentration was injected onto the sensor chips. rPV72 bound to the 2S-I peptide, but not to the 2S-I/3G peptide (Fig. 4 B), as expected from the result in Fig. 4 A. All of the deleted proteins, rPV72Δ3, rPV72Δ2,3, and rPV72Δ1,2,3, also bound to the 2S-I peptide, but not to the 2S-I/3G peptide (Fig. 4 B). This demonstrates that the deleted proteins also specifically recognize the RRE sequence of the 2S-I peptide as rPV72 does. These results indicated the N-terminal region of PV72 corresponding to rPV72Δ1,2,3 includes a ligand-binding site. We determined the kinetic parameters for the binding of each modified rPV72 to the 2S-I peptide by surface plasmon resonance. Each modified rPV72 was injected onto the 2S-I sensor chip to start the association reaction. Fig.5 A shows the association and dissociation curves obtained from the respective experiment with four different concentrations (0.07–2 μm) of each protein. The sensorgrams of rPV72Δ3, rPV72Δ2,3, and rPV72Δ1,2,3 exhibited more rapid association followed by more rapid dissociation after the injection was completed than did the sensorgram of rPV72. The kinetic constants of association and dissociation were calculated from the slopes of the curves, as shown in Fig. 5 B. In contrast to small differences of the k a values among the modified rPV72, large differences of the k dvalues were observed. The k d value of each of rPV72Δ3, rPV72Δ2,3, and rPV72Δ1,2,3 was 16–23-fold higher than the k d value of rPV72. The apparent equilibrium dissociation constant was determined from the ratio of these two kinetic constants (k d/k a). rPV72 has a high enough affinity for the 2S-I peptide (K D= 0.2 μm) to function as a receptor. TheK D values of each of rPV72Δ3, rPV72Δ2,3, and rPV72Δ1,2,3 were 10-, 19-, and 21-fold higher than theK d value of rPV72, respectively. Therefore, the affinity of rPV72 for the ligand peptide is much higher than the affinities of the rPV72s lacking the EGF-like motifs. It seems likely that the EGF-like motifs play a role in stabilizing the receptor-ligand complex. The question is how the EGF-like motifs regulate the stability of the ligand binding of PV72. Previously, we found that the third EGF-like motif has a consensus sequence for Ca2+binding, while the first and second motifs do not have such a sequence (26Shimada T. Kuroyanagi M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1997; 38: 1414-1420Crossref PubMed Scopus (101) Google Scholar). Fig. 4 A shows that rPV72 was eluted from the 2S-I column by the addition of chelating agents. This implied that Ca2+ binding to the third EGF-like motif might be important for the ligand binding. To clarify the requirement of Ca2+, we performed an analysis of surface plasmon resonance with rPV72 in the presence of either Ca2+ or Mg2+. Fig.6 A (left) shows that the interaction between rPV72 and the ligand was observed in the presence of Ca2+, but not in the presence of Mg2+ instead of Ca2+. This result indicates that Ca2+ is required for PV72 to interact with the 2S-I peptide. Unexpectedly, however, rPV72Δ1,2,3, which lacks the EGF-like motifs, also showed a Ca2+-dependent interaction with the 2S-I peptide, but not a Mg2+-dependent interaction (Fig. 6 A, right), as rPV72 did. This result indicates that the N-terminal region corresponding to rPV72Δ1,2,3 has another Ca2+-binding site (s), although no consensus sequence for Ca2+ binding was found in the region. It should be noted that the affinity of PV72 (K D = 0.2 μm) was 20-fold stronger than that of rPV72Δ1,2,3 (K D = 4.2 μm). Thus, the Ca2+-binding to the EGF-like motif must be required for the high affinity of PV72 for the ligand. The next question raised is whether the Ca2+ binding causes a conformational change of PV72 that results in the higher affinity. To answer the question, we measured Ca2+-dependent changes in the fluorescence emission" @default.
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