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PHYSIOLOGIA PLANTARUM 120: 319–327. 2004 Printed in Denmark – all rights reserved Copyright # Physiologia Plantarum 2004 Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein Wenxian Suna,c, Zhan Dong Zhaob, Michael C. Harec, Marcia J. Kieliszewskib,c and Allan M. Showaltera,c,* a Department of Environmental and Plant Biology, Department of Chemistry and Biochemistry, c Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701-2979, USA *Corresponding author, e-mail: [email protected] b Received 21 March 2003; revised 13 June 2003 Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato (Lycopersicon esculentum, cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension- cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling. Introduction Arabinogalactan proteins (AGPs) are a class of hydroxyproline-rich glycoproteins and form a family of structurally complex proteoglycans that are widely distributed throughout the plant kingdom from bryophytes to angiosperms (Fincher et al. 1983, Showalter 1993, Nothnagel 1997). AGPs occur in all organs and tissues, predominantly in cell walls, plasma membranes (PMs), and intercellular spaces (Nothnagel 1997). The use of monoclonal antibodies to carbohydrate epitopes on AGPs and b-Yariv phenylglycosides provide evidence that some AGPs function in signalling and extracellular matrix interactions during plant growth and development, cell differentiation, and somatic embryogenesis (Nothnagel 1997, Showalter 2001). AGPs can be grouped into two broad classes designated ‘classical’ and ‘non-classical’ (Mau et al. 1995, Du et al. 1996). Classical AGPs consist of three distinct domains: an N-terminal signal sequence, a Hyp-, Ala-, Ser-, and Thr-rich domain, and a C-terminal hydrophobic domain. The C-terminal hydrophobic domain is presumably proteolytically removed and replaced by a glycosylphosphatidylinositol (GPI) lipid anchor, allowing for attachment to the PM. Indeed, the C-terminal hydrophobic domains predicted from cDNAs encoding two Abbreviations – AGPs, arabinogalactan proteins; ELISAs, enzyme-linked immunosorbant assays; ESI-MS, electrospray ionization mass spectrometer; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; LC-MS, liquid chromatography- mass spectrometer; PI-PLC, phosphatidylinositol-specific phospholipase C; PM, plasma membrane. Physiol. Plant. 120, 2004 319 classical AGPs (NaAGP1 and PcAGP1) were absent in the mature proteins and replaced with a moiety containing ethanolamine, inositol, glucosamine and mannose (Youl et al. 1998). Oxley and Bacic (1999) went on to determine the detailed structure of this GPI anchor in PM-bound pear AGPs. They found that this structure was similar to GPI anchors found in animals, protozoa, and yeast and that it contained a ceramide lipid (Fig. 1). In addition, rose AGPs were shown to carry a ceramide class GPI lipid anchor based upon a combination of techniques including reverse phase HPLC, treatment with phosphatidylinositol-specific phospholipase C (PIPLC), and chemical structural analysis (Svetek et al. 1999). Using two-dimensional SDS-PAGE, Western blotting with AGP antibodies and PI-PLC, Sherrier et al. (1999) also demonstrated the presence of PM-bound AGPs in Arabidopsis. Recent analysis of the Arabidopsis genome for putative AGPs show that there are a large number of putative AGPs (approximately 47), many of which are predicted to be GPI anchored (approximately 35) (Schultz et al. 2002). Similarly, analysis of the Arabidopsis genome for putative GPI-anchored proteins showed that there are a large number of putative-GPI anchored proteins (approximately 210), 40% of which are predicted to be AGPs or to have AGP modules within them (Borner et al. 2002). LeAGP-1 is a major AGP in tomato cell cultures and plants, and represents one of the best-characterized AGPs to date. Its cDNA and genomic sequences are known and predict a classical AGP with four distinct regions: an N-terminal signal sequence for secretion, a central hydroxyproline/proline-rich region interrupted by a short lysine-rich basic region, and a hydrophobic C-terminal sequence identified as a putative GPI-anchor addition sequence (Pogson and Davies 1995, Li and Showalter 1996). The pattern of LeAGP-1 expression in cultured cells and plants on the mRNA level as well as the protein level is known, the later analysis being facilitated with a LeAGP-1 specific antibody (Li and Showalter 1996, Gao et al. 1999, Gao and Showalter 2000, Lu et al. 2001). The LeAGP-1 core protein was purified following deglycosylation and characterized biochemically in terms of amino acid composition, amino acid sequence, and SDS-PAGE/Western blot analysis (Gao et al. 1999). Most recently, native (i.e. glycosylated) LeAGP-1 was purified and its carbohydrate moiety analysed (Zhao et al. 2002). Based on this information, LeAGP-1 is hypothesized to be a classic AGP localized to the PM via a GPI anchor (Schultz et al. 1998, Gao et al. 1999). However, most of the biochemical characterization work to date on LeAGP-1, and indeed on most other AGPs, involves isolation from cell culture media where AGPs accumulate in large quantities, and not from PMs, which represent a more limited, transient, and technically challenging site of accumulation. Here we present several experiments that support the hypothesis that LeAGP-1 is localized to the PM via a glycosylphosphatidylinositol (GPI) anchor and discuss the functional implications for such a scenario. Materials and methods Isolation of tomato protoplasts and immunolocalization of LeAGP-1 Cotyledons from 10-day-old tomato (Lycopersicon esculentum, cv. UC82B) seedlings were collected, cut into pieces, and incubated with 1% (w/v) cellulase (Sigma, St. Louis, MO, USA) and 0.5% (w/v) pectinase (Sigma) on an orbital shaker for at least 4 h. Protoplasts were tracked with 0.01% (w/v) Fluorescent Brightener 28 (Calcofluor white M2R) (Sigma). Protoplasts were collected and fixed in 2% (w/v) paraformaldehyde and 1% glutaraldehyde (v/v) in 50 mM citrate-phosphate buffer (pH 7.4) for 2 h at 4 C, and then mounted onto poly-Llysine covered slides for immunolocalization of LeAGP-1 at the light microscope level using the PAP (i.e. LeAGP-1) antibody and FITC-conjugated sheep anti-rabbit IgG antibody as described previously (Gao et al. 1999). Transgenic tobacco cell lines expressing GFP-LeAGP-1 fusion proteins and fluorescence microscopy Transgenic tobacco (Nicotiana tabacum BY-2) cell lines expressing tomato GFP-LeAGP-1 fusion protein and a variant fusion protein lacking the C-terminal hydrophobic region of LeAGP-1, named GFP-LeAGP-1DC, were produced as described previously and utilized in this Fig. 1. GPI anchor structure for plasma membrane-bound AGPs. This chemical structure was determined for a pear PM-bound AGP (Oxley and Bacic 1999). The phosphoceramide lipid is embedded in the outer leaflet of the PM and is attached to the core oligosaccharide, which is in turn attached to the C-terminal amino acid residue (i.e. the o residue) of the AGP. The core oligosaccharide may contain a partial b-galactosyl substitution (*) on the core oligosaccharide. Potential sites of cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) and D (PI-PLD) are also indicated. Note that this structure is not drawn to scale. Et, ethanolamine; Man, mannose; Gal, galactose; GlcN, N-acetylglucosamine. 320 Physiol. Plant. 120, 2004 study (Zhao et al. 2002). Specifically, an enhanced version of GFP (i.e. EGFP) was used in these studies (Clontech, Palo Alto, CA, USA). In order to visualize the fusion proteins in transgenic suspension-cultured cells, fluorescence microscopy was performed using a Molecular Dynamics Sarastro 2000 confocal laser-scanning fluorescence microscope equipped with a FITC filter set consisting of a 488-nm laser wave length filter, a 510-nm primary beam splitter and a 510-nm barrier filter. Data were captured by Molecular Dynamics Image Space software. In some experiments, the tobacco cells were plasmolysed in 0–4% (w/v) NaCl solutions for 10 min before microscopic examination. Isolation of plasma membranes from tomato and transgenic tobacco suspension-cultured cells PM vesicles were isolated and purified following the procedures of Komalavilas et al. (1991) with minor modifications. Tomato cv. Bonnie Best cells and transgenic tobacco cells expressing GFP-LeAGP-1 or GFPLeAGP-1DC were harvested for PM isolation 10 days after subculture. Cells were washed thoroughly with distilled water, and homogenized with a model PTA 20S Polytron (Brinkmann, Westburg, NY, USA) using three 20-s bursts in a homogenizing buffer consisting of 50 mM Tris-HCl, 10 mM KCl, 1 mM EDTA, 0.1 mM MgCl2 and 8% (w/v) sucrose and 1 mM phenylmethylsulphonylfluoride (pH 7.5). Cell debris was removed by filtering and centrifugation, and the supernatant was ultracentrifuged for 45 min at 100 000 g to collect microsomal membranes. Pellets were suspended in a buffer (0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate [pH 7.8]). PM vesicles were purified by aqueous two-phase partitioning (Larsson et al. 1987, Komalavilas et al. 1991). Isolation of AGPs from PM vesicles AGPs were extracted from PM vesicles by incubating with 1% (w/w) Triton X-100 overnight at 4 C. The detergent-treated PM vesicles were centrifuged for 1 h at 100 000 g at 4 C. Supernatants were incubated with (b-D-galactosyl)3 Yariv reagent to precipitate and purify AGPs as previously described (Gao et al. 1999). Alternatively, absolute ethanol was added to the supernatant to a final concentration of 80% and incubated overnight at 4 C. The precipitating AGPs were collected by centrifugation, washed twice with ethanol, and then air-dried. Anhydrous hydrogen fluoride (HF) deglycosylation of AGPs and Reverse phase (RP)-HPLC of deglycosylated AGPs Anhydrous HF deglycosylation was carried out as previously described by Kieliszewski et al. (1994). Briefly, anhydrous HF was added to completely dried tomato PM-AGPs (PM-AGPs) at a concentration of 20 mg ml1 AGP and incubated for 1 h at 4 C, with occasional Physiol. Plant. 120, 2004 agitation. The reaction was quenched by the addition of ice-cold distilled water to a final HF concentration of 10%, dialysed against distilled water, and lyophilized. Deglycosylated AGPs (0.5 mg) were dissolved in a small amount of dH2O and spun briefly at 12 000 g. The supernatant was subjected to reverse-phase HPLC by injection into a Hamilton PRP-1 column, using gradient elution with 0.1% trifluoroacetic acid (TFA) and 80% acetonitrile in 0.1% TFA (Gao et al. 1999, Zhao et al. 2002). Western blot and enzyme-linked immunosorbent assays (ELISAs) Tomato PM-AGPs were examined with a LeAGP-1 specific antibody (PAP antibody), while PM-AGPs from transgenic tobacco cell lines were examined using an anti-GFP antibody (Clontech, Palo Alto, CA, USA) in order to detect GFP-LeAGP-1 fusion proteins. Quantification of proteins was accomplished with a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA). Western blotting was conducted as described previously (Gao et al. 1999). ELISAs were performed according to a standard protocol using alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma) as the secondary antibody (Harlow and Lane 1988). Ethanolamine analysis of GFP-LeAGP-1 and GFP-LeAGP-1DC GFP-LeAGP-1 and GFP-LeAGP-1DC were purified from transgenic tobacco cell culture media and treated with chymotrypsin to remove GFP as previously described (Zhao et al. 2002). The resulting LeAGP-1 and LeAGP-1DC glycoproteins were deglycosylated with HF as described above and then hydrolysed in 6 N HCl under vacuum at 110 C for 18 h. Samples were dried under vacuum, washed with ethanol, water, triethylamine (2:2:1), and redried under vacuum. Samples, as well as an ethanolamine standard, were derivatized with AQC and analysed by reverse phase HPLC (Waters AccQ-Tag C18 column, 150 4.6 mm i.d) using the gradient recommended by Waters for analysing collagen hydrolysates (Crimmins and Cherian 1997, Van Wandelen and Cohen 1997). Fluorescence was monitored by flow-through detection using a Hewlett-Packard 1100 series fluorometer (excitation ¼ 250 nm; emission ¼ 395 nm). The reversed phase peak corresponding to AQC-derivatized ethanolamine (34 min elution time) and a 34 min peak from the AQC-derivatized LeAGP-1 hydrolysate were collected for further analysis by electrospray ionization mass spectrometry. Although the control (LeAGP-1DC) lacked a fluorescent peak at 34 min (indicating the control lacked ethanolamine), the eluant from the column at 34 min was nevertheless collected for further analysis. The 34 min Waters AccQ-Tag reversed phase fractions described above were injected into a Vydac reverse phase C18 column (1 50 mm i.d) at a flow rate of 0.75 ml min1. Buffer A was 0.02% (v/v) TFA in water. Buffer B was 95% (v/v) aqueous 321 acetonitrile. The gradient went from 1 to 15% B in 15 min, then 15–50% B in 10 min. The Vydac column effluent was fed directly into an electrospray ionization mass spectrometer (ESI-MS). Liquid chromatography-MS (LC-MS) spectra were acquired using electrospray ionization on a Bruker Esquire ion trap instrument operating in the positive ion mode. The electrospray capillary was set at 4 kV. The sample flow was nebulized by nitrogen gas at 40 psi and dried using a countercurrent flow of nitrogen at 7 l min1 and 300 C. Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes. Results Immunolocalization of LeAGP-1 on tomato plasma membranes Protoplasts were isolated from tomato cotyledons using cellulase and pectinase. The PAP antibody (a LeAGP-1specific antibody) and preimmune serum (as a control) were used to immunolocalize LeAGP-1 to the PM of these protoplasts (Fig. 2A). Pre-immune controls showed no background immunofluorescence (Fig. 2B). LeAGP-1 present in tomato plasma membrane vesicles To further verify the presence of LeAGP-1 at the PM, biochemical separation strategies were used for purification of LeAGP-1 from tomato PM vesicles. Tomato suspension-cultured cells were collected and homogenized in a homogenizing buffer. Cell debris was separated from microsomal membranes through filtering and centrifugation. PMs were purified from other microsomal membranes by aqueous two-phase partitioning. AGPs were fully extracted from PM with 1% Triton X100 overnight and precipitated with (b-D-galactosyl)3 Yariv reagent. PM-AGPs were deglycosylated with anhydrous hydrogen fluoride, and the resulting protein backbones were fractionated by RP-HPLC (Fig. 3A). A A B Fig. 2. Immunolocalization of LeAGP-1 to the plasma membrane of a tomato cotyledon protoplast. (A) Tomato protoplast treated with the LeAGP-1 antibody/FITC-labelled secondary antibody. (B) Tomato protoplast treated with preimmune serum/FITC-labelled secondary antibody as a control. Bars ¼ 10 mm. Two major peak fractions (PM-1 and PM-3) and three minor peak fractions (PM-2, PM-4, and PM-5) were collected and subjected to further analysis. Analysis of the five peak fractions using the LeAGP-1 antibody by ELISAs (data not shown) and by Western blotting (Fig. 3B) demonstrated that LeAGP-1 is only present in the PM-3 fraction as a deglycosylated protein with an apparent molecular weight of 48 kDa. Fluorescence microscopy of transgenic tobacco cells expressing GFP-LeAGP-1 and GFP-LeAGP-1DC fusion proteins Tomato LeAGP-1 gene constructions with and without the C-terminal hydrophobic region were expressed as fusion proteins with GFP in transgenic tobacco suspensioncultured cells as previously described (Zhao et al. 2002). Preliminary results indicated that GFP-LeAGP-1 was present on the cell surface of these transgenic tobacco cells and in PMs and Hechtian strands of plasmolysed cells. This was substantiated and examined in more detail using the two cell lines, respectively, expressing GFPLeAGP-1 and GFP-LeAGP-1DC fusion proteins and treating them with various concentrations of NaCl B Fig. 3. Biochemical separation and identification of LeAGP-1 from plasma membrane vesicles isolated from tomato suspension-cultured cells. (A) Reverse-phase HPLC separation of (b-D-galactosyl)3 Yariv reagent-precipitable, HFdeglycosylated AGPs from tomato plasma membrane vesicles. (B) Western blot analysis of HPLC peak PM-3 using the LeAGP-1 antibody/alkaline phosphataselinked secondary antibody. Molecular masses (kDa) of the protein size standards are shown along with the calculated molecular mass of the reacting protein (i.e. deglycosylated LeAGP-1). 322 Physiol. Plant. 120, 2004 ranging from 0 to 4% (w/v) (Fig. 4). Cells expressing GFP-LeAGP-1 and not treated with NaCl (0% NaCl) demonstrated green fluorescence at the cell surface. With increasing percentages of salt treatment, plasmolysis was observed and green fluorescence was increasingly observed at the PM and in Hechtian strands, which are connections between the PM and cell wall (Fig. 4B-F). Cells expressing GFP-LeAGP-1DC also demonstrated cell surface fluorescence; however, this fluorescence was uniformly distributed in the cell wall–PM interface and did not clearly discern the PM or Hechtian threads (Fig. 4G). Moreover, cells expressing GFP-LeAGP-1DC consistently secreted 6–10 times more fusion protein (i.e. 4.9–6.3 mg l1) into the culture media than cells expressing GFP-LeAGP-1 (0.46–1.1 mg l1). GFP-LeAGP-1 present in plasma membrane vesicles of transgenic tobacco cells expressing GFP-LeAGP-1, but not GFP-LeAGP-1DC Since the C-terminal hydrophobic domain of LeAGP-1 contains a putative GPI anchor addition site, and transgenic GFP-LeAGP-1DC is apparently not tightly associated with the PM, it was hypothesized that GFPLeAGP-1 should be anchored to the PM, while GFPLeAGP-1DC should not. In order to test this hypothesis, A GFP-LeAGP-1: GFP-LeAGP-1∆C: Signal peptide domain GFP domain Lys-rich domain C-terminal hydrophobic domain B C PM/CW D F Physiol. Plant. 120, 2004 E PM H G “Classical” AGP domain Fig. 4. Domain organization and expression of GFP-LeAGP-1 fusion constructs in transgenic tobacco cell suspension cultures. (A) Organization of the GFPLeAGP-1 and GFP-LeAGP-1DC fusion proteins expressed in transgenic tobacco cells. GFPLeAGP-1 consists of the N-terminal LeAGP-1 signal peptide, an inserted GFP domain, a central classical AGP domain sandwiching a highly basic Lysrich domain, and a C-terminal hydrophobic domain; GFPLeAGP-1DC has the same organization except the C-terminal hydrophobic domain, presumably essential for GPIanchor addition, is absent. (B-G) Detection of GFP fluorescence in tobacco cells expressing GFPLeAGP-1 following treatment with culture media supplemented with 0% NaCl (B), 1% NaCl (C), 2% NaCl (D), 3% NaCl (E), and 4% NaCl (F) for 10 min. Detection of GFP fluorescence in tobacco cells expressing GFPLeAGP-1DC following treatment with culture media supplemented with 4% NaCl (G). Hechtian strands are clearly visible in plasmolysed tobacco cells expressing GFP-LeAGP-1, particularly following treatment with 2–4% NaCl. PM, plasma membrane; CW, cell wall; H, Hechtian threads. Bars ¼ 10 mm. 323 total AGPs were isolated from PM vesicles of transgenic tobacco cells expressing GFP-LeAGP-1 and GFPLeAGP-1DC. Western blotting analysis using a GFP antibody indicated that GFP-LeAGP-1 was indeed present in these PM preparations, while GFP-LeAGP-1DC was not (Fig. 5A). Moreover, some of the 28 kDa GFP was apparently cleaved from the higher molecular weight fusion protein. Such cleavage can largely be attributed to heating the protein samples prior to gel loading, since GFP-LeAGP-1 samples loaded on a gel without heating had considerably less of the 28 kDa GFP protein released from the fusion protein compared to that of the heated sample (Fig. 5B). Ethanolamine analysis of the GFP-LeAGP-1 fusion protein In order to provide evidence for a GPI anchor on LeAGP-1, GFP-LeAGP-1 was purified from transgenic tobacco cell culture media, as was GFP-LeAGP-1DC, and analysed for the presence of ethanolamine, a known substituent attached to the C-terminal residue of GPIanchored proteins even after their secretion. Indeed, reversed phase fractionation of 6-aminoquinolyl-Nhydroxysuccinimidylcarbamate (AQC)-derivatized LeAGP-1 hydrolysate yielded a small peak that eluted at 34 min, coincident with AQC-derivatized ethanolamine standard, whereas the control, LeAGP-1DC, did not (Fig. 6). Further fractionation of the 34 min peak on a Vydac column yielded a peak eluting at 7 min. The ethanolamine A Discussion The association of AGPs with the PMs of plant cells is well documented in the scientific literature; however, only in the past 5 years have researchers elucidated the nature of this association. Certain AGPs are now known to be attached to the PM via a GPI lipid anchor (Fig. 1). Since GPI anchors require a C-terminal consensus sequence for their addition, data mining of gene sequences can be performed to reveal a large number of putative GPI-anchored AGP sequences. Using two complementary approaches, two research groups performed such analyses. Specifically, Borner et al. (2002) used a search algorithm to first identify 210 putative GPI anchored proteins in the Arabidopsis genome and then found that 40% of these proteins are predicted to be AGPs or include AGP modules. In contrast, Schultz B Fig. 5. Identification of GFP-LeAGP-1 from plasma membrane vesicles isolated from transgenic tobacco suspension-cultured cells. (A) SDS-PAGE/Western blot analysis of plasma membrane (PM) proteins from transgenic tobacco cell cultures expressing GFPLeAGP-1 or GFP-LeAGP-1DC fusion proteins using a GFP antibody/FITC-labelled secondary antibody. Lanes 1 and 3: 10 mg and 5 mg total PM protein from transgenic tobacco cells expressing GFP-LeAGP-1; lanes 2 and 4: 10 mg and 5 mg total PM protein from transgenic tobacco cells expressing GFP-LeAGP-1DC. B. SDSPAGE/Western blot analysis of GFP-LeAGP-1 treated with (1) or without (–) heat (100 C for 5 min) prior to gel loading. The GFP antibody/alkaline phosphatase-labelled secondary antibody was used for detection. 324 standard also eluted from the Vydac column at 7 min. The ethanolamine standard, which eluted at 7 min on the Vydac column, had a molecular mass of 231 Da, as did the 7 min Vydac peak from LeAGP-1. Fig. 6. Identification of AQC-derived ethanolamine (EA-AQC) prepared from (A) an ethanolamine standard, or from (B) the acid hydrolysate of HF-deglycosylated LeAGP-1. Samples were fractionated by reverse phase column chromatography and the peaks eluting at 34 min were collected for atomic mass determination by LC-MS. The additional peaks occurring in panels A and B correspond to the remaining AQC-derived amino acids in the LeAGP-1 hydrolysate (B) and by-products of the AQC derivatization reaction (A and B). The fluorescence (LU, luminescence units) was measured using 250 nm and 395 nm as the excitation and emission wavelengths, respectively. Physiol. Plant. 120, 2004 et al. (2002) used a search algorithm to first identify 47 putative AGP genes from the Arabidopsis genome and subsequently predicted that 35 of these genes encode GPI-anchored products. These complementary data mining studies, coupled with other biochemical analyses of PM bound AGPs, indicate that AGPs are encoded by a large gene family with a substantial number of these AGPs predicted to be GPI-anchored to the outer leaflet of the PM where they likely constitute a major surface decoration in the form of arabinogalactan glycomodules. While several AGPs are predicted to be GPI anchored to the PM, only a few AGPs are actually known to contain this anchor based on biochemical analysis (Youl et al. 1998, Oxley and Bacic 1999, Sherrier et al. 1999, Svetek et al. 1999). LeAGP-1, a major AGP present in tomato cultured cells and plants, is hypothesized to be GPI-anchored to the PM based on the presence of a C-terminal consensus sequence for GPI addition, as well as immunolocalization at the cell surface, and here immunochemical and biochemical evidence is presented to support this hypothesis. This work was undertaken, not simply to support this hypothesis, but to provide additional information on LeAGP-1 to supplement the wealth of structural information on this novel AGP with its characteristic Lys-rich subdomain and thereby facilitate its functional identification. Five lines of experimental evidence are presented here in support of this hypothesis. First, LeAGP-1 can be immunolocalized to the PM in tomato cotyledon protoplasts (Fig. 2). Years earlier, Larkin (1978), working with several plant species, demonstrated that AGPs are at the surface of protoplasts by treating protoplasts with Yariv reagent and causing their agglutination. Similarly, antibodies developed against carbohydrate epitopes on AGPs also show PM localization, but such epitopes are shared by multiple AGPs (reviewed in Showalter 2001). In contrast to Yariv reagent and AGP antibodies directed against carbohydrate epitopes, which react with multiple AGPs, the LeAGP-1 antibody used here is highly selective and allows for specific identification of LeAGP-1 (Gao et al. 1999). Second, LeAGP-1 is present in PM vesicles produced from cultured tomato cells (Fig. 3). This evidence was ascertained by examining deglycosylated AGPs (i.e. the AGP core proteins) obtained from these vesicles, again using the LeAGP-1 antibody. Notably, other AGPs are apparently present in these vesicles, and furthermore the HPLC profile of the eluting core proteins is similar to that previously observed for the culture media (Gao et al. 1999). Thus, as many as five different AGP core proteins may be present with LeAGP-1 (peak PM-3) and the AGP represented in PM-1 being the most abundant. The LeAGP-1 core protein has an apparent molecular weight of 48 kDa, again identical to that previously observed for the LeAGP-1 core protein isolated from culture media (Gao et al. 1999). It should be noted, however, that the LeAGP-1 gene predicted a core protein (sans signal and C-terminal hydrophobic peptides) with a molecular weight of approximately 16 kDa. This indicated Physiol. Plant. 120, 2004 either a crosslinked dimer/trimer or anomalous gel migration of this Hyp-rich protein. Analysis of deglycoslyated LeAGP-1 by MALDI-TOF mass spectrometry yielded a mass of approximately 16 kDa (unpublished results) consistent with the later alternative. Third, GFP-LeAGP-1 fusion proteins were expressed in transgenic tobacco cells and demonstrated GFP fluorescence at the PM and its extensions (i.e. Hechtian strands) following plasmolysis. While GFP fluorescence is seen at the cell surface (i.e. at the CW/PM interface) in turgid cells, it was only after plasmolysis that localization to the PM and Hecht’s threads was clearly observed (Fig. 4). The presence of LeAGP-1 in Hechtian strands may indicate roles for this AGP in these poorly characterized entities, which serve as attachment sites between the PM and CW. In contrast, the truncated version of LeAGP-1, lacking the C-terminal hydrophobic domain, did not clearly display PM or Hechtian thread fluorescence, but was still present in the CW/PM interface in plasmolysed cells and indeed accumulated to much higher levels in the culture media than the full length LeAGP-1 fusion product. These data are consistent with the prediction that the C-terminal domain contains a consensus signal for GPI addition, which serves to retain LeAGP-1 at the PM. Fourth, the GFP-LeAGP-1 fusion protein is present in PM vesicles produced from the transgenic tobacco cultured cells (Fig. 5). However, the truncated version of LeAGP-1 lacking the C-terminal hydrophobic domain did not appear in these vesicle preparations, consistent with the predicted role of the C-terminus in signalling addition of a GPI membrane anchor and with the corresponding GFP localization data that failed to demonstrate clear PM fluorescence (Fig. 4G). Specifically, two bands are observed in the Western blot analysis of PM vesicle preparations using the GFP antibodies. One high molecular weight band (approximately 98–120 kDa) represents the glycosylated fusion protein. This product was previously isolated and characterized from the culture media and demonstrated to be glycosylated with large amounts of arabinose and galactose and lesser amounts of glucuronic acid and rhamnose (Zhao et al. 2002). The broadness of this high molecular weight band reflects the heterogeneous nature of core protein glycosylation. The smaller product was 28 kDa and represents GFP which is cleaved from the fusion protein in block upon heat treatment, perhaps through peptide bond cleavage via an N!O acyl shift. (Xu et al. 1999). Related and complementary to the above information, Takos et al. (2000) fused a Clostridium thermocellum endoglucanase E reporter gene (celE0 ) to the C-terminal hydrophobic domain of LeAGP-1. They expressed these constructs in tobacco protoplasts and demonstrated that the C-terminal hydrophobic domain of LeAGP-1 directed the addition of a GPI anchor to the endoglucanase reporter protein based on sensitivity to PI-PLC digestion. Fifth, ethanolamine was identified in the GFPLeAGP-1 fusion protein secreted into the transgenic tobacco cell culture media. Ethanolamine is added to 325 the so-called C-terminal o residue of GPI-anchored proteins during anchor addition and remains with the protein even upon release from the PM (Udenfriend and Kodukula 1995, Schultz et al. 1998). The identification of ethanolamine indicates LeAGP-1 indeed contains a GPI anchor. The truncated version of LeAGP-1 lacking the C-terminal hydrophobic domain, however, did not contain ethanolamine, consistent with its predicted inability to carry out the C-terminal proteolytic processing required for anchor addition. It is now clear that LeAGP-1 is localized to the PM via a GPI anchor and is subsequently processed for release to the cell wall, extracellular space, and culture media. Indeed, previous biochemical and immunolocalization studies on LeAGP-1 in tomato as well as GFP-LeAGP-1 in transgenic tobacco have documented LeAGP-1 in these extracellular sites of accumulation (Gao et al. 1999, Gao and Showalter 2000, Zhao et al. 2002). In particular, it is worth noting that LeAGP-1 as well as other tomato AGPs appear to be present in both the PM and in the culture media based upon their similar HPLC core protein profiles as mentioned above. Such processing most likely involves the action of PI-PL C or D (Fig. 1). Oxley and Bacic (1999) suggested that PI-PL D is likely to operate, with or without prior PI-PL C action, since a secreted pear AGP (PcAGP1) lacks the phosphoceramide moiety. While plants contain several phospholipases, only one report documents the existence of a plant PL with the ability to cleave GPI anchors, namely PI-PL C from peanut (Butikofer and Brodbeck 1993). Several functional scenarios are envisioned for LeAGP-1, which are in part based on the transgenic expression of LeAGP-1 in tobacco. This transgenic work likely reflects the natural situation in tomato, since tobacco and tomato are close evolutionary relatives, both species express LeAGP-1 (the orthologous gene in tobacco is called NaAGP4), and transgenic LeAGP-1 is processed and modified in tobacco similar to that of endogenous LeAGP-1 in tomato (Gilson et al. 2001, Zhao et al. 2002). Thus, given that LeAGP-1 is GPI anchored and released from the PM, the following functional scenarios are envisioned for this AGP. First, LeAGP-1 may serve as a marker of cellular identity. In this context, the arabinogalactan polysaccharides that decorate noncontiguous Hyp, and the oligoarabinosides attached to contiguous Hyp residues of LeAGP-1 would constitute part of a glycocalyx containing information-rich, molecular surface markers (Zhao et al. 2002). The ability to shed or turnover such markers is inherently associated with GPI-anchored proteins, and could be regulated in response to cellular, developmental, or environmental cues where remodelling of the cell surface is required (Nosjean et al. 1997). Second, LeAGP-1 may serve as a cell membrane receptor or mediator of cellular signalling. Although LeAGP-1 lacks a cytoplasmic domain, it may associate with other membrane proteins, such as transmembrane proteins, which do contain cytoplasmic domains capable of initiating an intracellular signalling cascade. For example, LeAGP-1 may function analo326 gously to certain GPI-anchored heparan sulfate proteoglycans which serve to bind ligands for delivery to a membrane receptor tied into a signalling network (Schlessinger et al. 1995). It is also possible that the GPI moiety of LeAGP-1 may serve as a signal itself following release of LeAGP-1 from the PM, since phosphatidyl inositol, inositol phosphoglycan and ceramides, breakdown products of the GPI anchor, are all known intracellular signalling molecules (Jones and Varela-Nieto 1998). Third, LeAGP-1 may function as a linker between the PM and cell wall. While LeAGP-1 is clearly found in sites of PM-CW adhesion (i.e. Hecht’s threads) (Fig. 4F), it is also distributed throughout the PM. Thus, its distribution is not restricted to Hecht’s threads, leaving unanswered the questions of whether a punctuate distribution is required for suspected PM-CW adhesion molecules (Gens et al. 2000) and whether LeAGP-1 is simply ‘pulled’ into the Hecht’s strands by virtue of its apparent uniform distribution on the PM. Finally, these and any other functional scenarios should also be considered in light of the possibility that LeAGP-1, as well as other GPI-anchored proteins, may exist in lipid rafts. GPI-anchored proteins in mammalian cells are essentially universally targeted to lipid rafts, sphingolipidand cholesterol-rich PM microdomains, where they have proposed functions in polarized sorting, signal transduction, and regulation of cell-surface hydrolytic activity (Simon and Toomre 2000, Brown 2002). Testing of the above and other functional scenarios for LeAGP-1 and its Arabidopsis homologues is underway in the hope of elucidating the role of this well characterized, abundant, GPI-anchored PM AGP in plant growth and development. Acknowledgements – This work was supported by grants from the National Science Foundation (IBN-9727757 and IBN-0110413) to A.M.S and M.J.K. The authors thank Dr Li Tan and Dr Jianfeng Xu for technical assistance with HPLC and HF deglycosylation, Mr Jeff Thuma for confocal scanning microscopy, and Dr Li-Wen Wang and Mr Ming Chen for helpful advice. References Borner GHH, Sherrier DJ, Stevens TJ, Arkin IT, Dupree P (2002) Prediction of glycosylphosphatidylinositol (GPI) -anchored proteins in Arabidopsis: a genomic analysis. Plant Physiol 129: 486–499 Brown D (2002) Structure and function of membrane rafts. Int J Med Microbiol 291: 433–477 Butikofer P, Brodbeck U (1993) Partial purification and characterization of a (glycosyl) inositolphospholipid-specific phospholipase C from peanut. J Biol Chem 268: 17794–17802 Crimmins DL, Cherian R (1997) Increasing the sensitivity of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate amino acid analysis: a simple solution. Anal Biochem 244: 407–410 Du H, Simpson RJ, Clarke AE, Bacic A (1996) Molecular characterization of a stigma-specific gene encoding an arabinogalactanprotein (AGP) from Nicotiana alata. Plant J 9: 313–323 Fincher GB, Stone BA, Clarke AE (1983) Arabinogalactanproteins: structure, biosynthesis, and function. Ann Rev Plant Physiol 34: 47–70 Gao M, Kieliszewski MJ, Lamport DTA, Showalter AM (1999) Isolation, characterization and immunolocalization of a novel, Physiol. Plant. 120, 2004 modular tomato arabinogalactan-protein corresponding to the LeAGP-1 gene. Plant J 18: 43–55 Gao M, Showalter AM (2000) Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato. Planta 210: 865–874 Gens JS, Fujiti M, Pickard BG (2000) Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices. Protoplasma 212: 115–134 Gilson P, Gaspar YM, Oxley D, Youl JJ, Bacic A (2001) NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection in Nicotiana alata. Protoplasma 215: 128–139 Harlow E, Lane D (1988) Antibodies: a Laboratory Manual. Cold Spring Harbor Laboratory, New York, NY, pp. 564–565 Jones DR, Varela-Nieto I (1998) The role of glycosyl-phosphatidylinositol in signal transduction. Int J Biochem Cell Biol 30: 313–326 Kieliszewski MJ, Showalter AM, Leykam JF (1994) Potato lectin: a modular protein sharing sequence similarity with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors). Plant J 5: 849–861 Komalavilas P, Zhu J-K, Nothnagel EA (1991) Arabinogalactanproteins from the suspension culture medium and plasma membrane of rose cells. J Biol Chem 266: 15956–15965 Larkin PJ (1978) Plant protoplast agglutination by artificial carbohydrate antigens. J Cell Sci 30: 283–292 Larsson C, Widell S, Kjellbom P (1987) Preparation of high-purity plasma membranes. Meth Enzymol 148: 558–568 Li S-X, Showalter AM (1996) Cloning and developmental/stressregulated expression of a gene encoding a tomato arabinogalactan protein. Plant Mol Biol 32: 641–652 Lu H, Chen M, Showalter AM (2001) Developmental expression and pertubation of arabinogalactan-proteins during germination and seedling growth in tomato. Physiol Plant 112: 442–450 Mau SL, Chen CG, Pu ZY, Moritz RL, Simpson RJ, Bacic A, Clarke AE (1995) Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata. Plant J 8: 269–281 Nosjean O, Briolay A, Roux B (1997) Mammalian GPI proteins: sorting, membrane residence and functions. Biochimica Biophysica Acta 1331: 153–186 Nothnagel EA (1997) Proteoglycans and related components in plant cells. Int Rev Cytol 174: 195–291 Oxley D, Bacic A (1999) Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells. Proc Natl Acad Sci USA 96: 14246–14251 Pogson B, Davies C (1995) Characterization of a cDNA encoding the protein moiety of a putative arabinogalactan protein from Lycopersicon esculentum. Plant Mol Biol 28: 347–352 Schlessinger J, Lax I, Lemmon M (1995) Regulation of growth factor activation by proteoglycans: what is the role of the low affinity receptors? Cell 83: 357–360 Schultz C, Gilson P, Oxley D, Youl J, Bacic A (1998) GPI-anchors on arabinogalactan-proteins: implications for signalling in plants. Trends Plant Sci 3: 426–431 Schultz CJ, Rumsewicz MP, Johnson KL, Jones BJ, Gaspar YM, Bacic A (2002) Using genomic resources to guide research directions. The arabinogalactan protein gene family as a test case. Plant Physiol 129: 1448–1463 Sherrier DJ, Prime TA, Dupree P (1999) Glycosylphosphatidylinositol-anchored cell-surface proteins from Arabidopsis. Electrophoresis 20: 2027–2035 Showalter AM (1993) Structure and function of plant cell wall proteins. Plant Cell 5: 9–23 Showalter AM (2001) Arabinogalactan proteins: structure, expression and function. Cell Mol Life Sci 58: 1399–1417 Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nature Rev: Mol Cell Biol 1: 31–41 Svetek J, Yadav MP, Nothnagel EA (1999) Presence of a glycosylphosphatidylinositol lipid anchor on rose arabinogalactan proteins. J Biol Chem 274: 14724–14733 Takos AM, Dry IB, Soole KL (2000) Glycosyl-phosphatidylinositolanchor addition signals are processed in Nicotiana tabacum. Plant J 21: 43–52 Udenfriend S, Kodukula K (1995) Prediction of o site in nascent precursor of glycosylphosphatidylinositol protein. Meth Enzymol 250: 571–582 Van Wandelen C, Cohen SA (1997) Amino acid analysis of unusual and complex samples based on 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate derivatization. J Chromatogr A763: 11–22 Xu Q, Buckley D, Guan C, Guo H-C (1999) Structural insights into the mechanism of intramolecular proteolysis. Cell 98: 651–661 Youl JJ, Bacic A, Oxley D (1998) Arabinogalactan-proteins from Nicotiana alata and Pyrus communis contain glycosylphosphatidylinositol membrane anchors. Proc Natl Acad Sci USA 95: 7921–7926 Zhao ZD, Tan L, Showalter AM, Lamport DTA, Kieliszewski MJ (2002) Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis. Plant J 31: 431–444 Edited by D. Van Der Straeten Physiol. Plant. 120, 2004 327