Planta
Planta (1988) 173:482-489
9 Springer-Verlag 1988
Wheat-germ agglutinin is synthesized as a glycosylated precursor Michael A. Mansfield 1, Willy J. Peumans 2 and Natasha V. Raikhel i , MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824-1312, USA, and 2 Laboratorium voor Plantenbiochimie, Katholieke Universitat Leuven, Kardinaal Mercierlaan 92, B-3030 Leuven, Belgium
The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [35S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADHcytochrome c reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles. Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with Mr 21000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of Mr 23000 and the mature monomer of Mr 18000. When [aH]mannose was used in labeling studies, only the polypeptide of Mr 23000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of Mr 20000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the Abstract.
* To whom correspondence should be addressed Abbreviations: ER=endoplasmic reticulum; IgG=immunoglobulin G; Mr = relative molecular mass; poly(A) +RNA = polyadenylated RNA; SDS-PAGE=sodium dodecyl sulfatepolyacrylamide gel electrophoresis; WGA = wheat-germ agglutinin
carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745-6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide. K e y w o r d s : Glycoprotein precursor - Lectin - Protein processing - Triticum (wheat germ agglutinin) - Wheat germ agglutinin.
Introduction
At least three classes of vacuolar proteins are found in higher plants: acid hydrolases, lectins and storage proteins. Many of these proteins are synthesized as pre-pro-proteins on the rough endoplasmic reticulum (ER) and undergo cotranslational cleavage of the signal sequence in the lumen of the ER. The polypeptide may also be glycosylated with a glycan which is subject to further modification. In the Golgi apparatus, the polypeptides are packaged into vesicles that deliver them to the vacuoles, where further proteolytic cleavage may take place to yield the mature proteins (for a recent review, see Chrispeels 1984). The patterns of cleavage observed in different plant species are varied. Processing of rice glutelin, pea legumin, pea lectin, and 11S storage proteins of legumes involves conversion of one polypeptide to two subunits (see Higgins 1984 for review). Post-translational modification of a sulfur-rich protein from Brazil nut (Bertholletia excelsa) includes loss of the N-terminal domain (Sun et al. 1987). Loss of several amino acids from the carboxyl-terminal domain has been reported for pea lectin (Higgins et al. 1983) and for napin, a rapeseed storage protein (Crouch et al. 1983; Ericson et al. 1986). In the case of the lectin concanavalin A, posttranslational processing is necessary to generate a functional molecule. The polypeptide sequence un-
M.A. Mansfield et al. : Processing of wheat-germ agglutinin
dergoes several proteolytic cleavages and religation to yield the mature molecule (Bowles et al. 1986; Chrispeels et al. 1986). The biosynthesis of concanavalin A is of particular interest because pro-concanavalin A is glycosylated and the glycan is lost during processing (Herman et al. 1985). When glycosylation of pro-concanavalin A was prevented with tunicamycin, the unglycosylated precursor accumulated in the ER and Golgi (Faye and Chrispeels 1987). This phenomenon, however, may be unique to concanavalin A. Many vacuolar proteins are not glycoproteins (Higgins 1984) and preventing glycosylation of some proteins with tunicamycin did not affect their intracellular accumulation in protein bodies-vacuoles (Chrispeels et al. 1982; Bollini et al. 1983; Lord 1985). Wheat-germ agglutinin (WGA) is a lectin found in the embryos of wheat seeds (Mishkind et al. 1980). The maximum abundance of WGA is several micrograms per embryo (Mishkind et al. 1983), and WGA is retained or synthesized de novo many days after germination (Mishkind et al. 1980; Raikhel et al. 1984a). In wheat embryos, WGA is transported to vacuoles in specific regions of the coleoptile, radicle, coleorhiza, scutellum, and epiblast (Mishkind et al. 1982, 1983). Like many vacuolar proteins, WGA is processed during its transport to the vacuoles. The mature protein is a dimer consisting of two unglycosylated subunits of 171 amino acids each (Wright 1987). Accumulation of WGA in vacuoles implies passage through the ER. Recent analysis of a cDNA for WGA demonstrated the coding potential for a polypeptide extending 15 residues beyond the caroboxy-terminus of the mature protein (Raikhel and Wilkins 1987). This carboxylterminal sequence is more hydrophobic than the rest of the protein and contains a potential glycosylation site. These observations indicate that processing of W G A may be more complex than previously appreciated. In this study we show that pro-WGA is a glycosylated precursor and that the carboxyl-terminal end of the protein and glycan are lost during the transport process. Material and methods Plant material. Wheat (Triticum aestivum L. cv. Marshall; obtained from Minnesota Crop Improvement Association, St. Paul, USA) plants were grown in a growth chamber with a 16-h light period at 25~ and an 8-h dark period at 15~ C. Other growth conditions were as specified in Raikhel and Quatrano (1986). Unless otherwise indicated, experiments were carried out on developing embryos harvested at 20 d post-anthesis. Embryos were squeezed from the grains using forceps and washed free of liquid endosperm in distilled water.
483
Organelle analysis. In-vivo labeling, tissue homogenization, organelle isolation, agglutination assays, and gradient analysis were performed as described in Stinissen et al. (1984). Homogenization buffer A contained 100 mM 2-amino-2-(hydroxymethyl)-l,2-propanediol (Tris)-HCl, pH 7.8, and 12% (w/v) sucrose. Wheat-germ agglutinin in fractions eluted from Sepharose 4B or gradients was affinity-purified on immobilized Nacetylglucosamine (Selectin; Pierce Chemical Co., Rockford, Ill., USA) (Mishkind et al. 1980). Polyaerylamide gel electrophoresis. To optimize the resolution of WGA, proteins were first carboxyamidated (Raikhel et al. 1984a). The polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels (Laemmli 1970). Radioactively labeled proteins were visualized by fluorography with En3Hance (New England Nuclear, Boston, Mass., USA). Tissue fixation and immunoeytochemistry. All procedures, unless otherwise specified, were carried out at room temperature. Immunoreagents were centrifuged at 5000-g for 10 rain prior to use while all other solutions were sterilized by filtration. Embryos were collected and fixed in 3% glutaraldehyde in 25 mM Na-phosphate buffer (pH 7) for 1.5 h at room temperature, post-fixed in 2% OsO4 in the same buffer for 1.5 h, and then washed with additional buffer. Following dehydration in an ethanol series, the tissue was embedded in Spurr's resin (Polysciences, Warrington, Penn., USA). Thin sections were prepared on an Ultracut E microtome (Reichert-Jung, Buffalo, N.Y., USA) and mounted on parlodion-carbon coated gold grids (Polysciences). Immunogold labeling was performed on the grids. Sections were treated with saturated NaIO4 for 10 rain, washed in deionized water, treated with 0.1 N HC1 for 10 rain, and rinsed in deionized water (Craig and Goodchild 1984). Anti-WGA antibody (Mishkind et al. 1982) at a concentration of 30 ~tg.m1-1 in Tris-buffered saline containing 0.1% bovine serum albumin (TBS-BSA: 50raM Tris-HCl, pH 7.8, 150mM NaCI, 0.1% bovine serum albumin (Sigma), 0.05% polyoxyethylenesorbitan monolaurate (Tween-20; Sigma) was then applied for 12 h at 4~ C. After washing in TBS-BSA, the sections were incubated for 1 h with goat-anti-rabbit-immunoglobulinG (IgG) bound to colloidal gold (10 nm; Janssen Pharmaceutica, Beerse, Belgium) diluted 20-fold in TBS-BSA. After immunolabeling, the grids were treated with 1% glutaraldehyde in TBS for 10 rain, washed with deionized water, and then stained with 5% uranyl acetate and lead citrate. Controls included treatment with antiWGA antibody preadsorbed with purified WGA, and treatment with non-immune rabbit-IgG (Sigma) instead of antiWGA antibody. Thin section were examined on a JEOL (Tokyo, Japan) 100CXII transmission microscope. Isolation of WGA from in-vivo labeled embryos. A square of Parafilm (Fisher Scientific, Livonia, Mich., USA) was placed on top of water-saturated Blot Block paper (Schleicher and Schuell, Keene, N.H., USA) in a 100-ram Petri dish. Embryos were placed on the Parafihn in a 400-1xl drop of incubation solution (10-4 M ABA (Sigma) in sterile water). Exogenous ABA enhances synthesis of WGA in isolated embryos (Triplett and Quatrano 1982). After 2 h of preincubation, the solution was removed and replaced with fresh incubation solution containing 1.89 MBq L-[3~S]methionine (43000 GBq.mmol-1; New England Nuclear) or 1.89MBq D-[2,6-3H]mannose (2040 GBq-retool-1; Amersham Corp., Arlington Heights, IlL, USA). All incubations were carried out at room temperature. For the tunicamycin experiments, embryos were cut along the median longitudinal axis with a clean razor blade to facili-
484 tate uptake. The initial incubation solution contained 10 -4 M ABA, 0.5 M NaOH, and 0.5 mg-m1-1 tunicamycin (Sigma). For labeling, the incubation solution contained 10 4 M ABA, 0.5 mg-m1-1 tunicamycin and [35S]methionine as described above. Embryos were homogenized in 600 ~tl of 50 mM sodium acetate (pH 3.8), 0.1 M NaC1, containing 1 mM phenylmethylsulfonyl fluoride (Sigma). The homogenizer was washed with 600 gl of homogenization buffer. The homogenate was centrifuged at 16000.g for 10 min to remove debris, and (NH4)2SO 4 was added to the supernatant to 60% saturation. After 2 h at 4~ C, the precipitate was pelleted. The pelleted was resuspended in buffer B (0.05 M Tris-acetate, pH 5, 0.1 M NaC1) and passed over a N-acetylglucosamine affinity microcolumn (Selectin I; Pierce Chemical Co.) with a bed volume of approx. 75 gl. After washing the column with five 100-gl aliquots of buffer B, bound WGA was eluted with sequential 100 gl and 50-l.tl aliquots of 0.1 M N-acetylglucosamine. The aliquots were combined and lyophilized. The pellet was resuspended in Laemmli sample buffer (Laemmli 1970). For immunoprecipitation, the original supernatant was neutralized with 0.1 volumes of 1 M Tris-HCl (pH 7.8). Nacetylglucosamine was added from a 1 M stock to give a final concentration of 0.1 M. To remove nonspecifically binding proteins, 30 gl of Pansorbin (Calbiochem, San Diego, Cal., USA) cells, pre-washed with immunoprecipitation buffer (20 mM Tris-HC1, pH 7.4, 0.15 M NaC1, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate), were added to the sample. After 25 rain at 4~ C, the Pansorbin cells were removed by centrifugation at 16000.g for 2 rain. The supernatant was then mixed with a rabbit polyclonal serum developed against WGA (Mishkind et al. 1982) and incubated overnight at 4 ~ C. To isolate the WGA-antibody complexes, 50 gl of Pansorbin were added. After incubation at 4 ~ C for 6 h, the Pansorbin cells were pelleted by centrifugation at 16000.g for 2 rain. The supernatant was removed, and the pellet was resuspended in 750 gl of immunoprecipitation buffer. The Pansorbin cells were once again collected by centrifugation. This washing step was repeated three times with immunoprecipitation buffer and once with 50 mM Tris-HC1 (pH 7.4). The final pellet was resuspended in 25 gl of Laemmli sample buffer and heated at 100~ C for 2 rain. The Pansorbin cells were pelleted by centrifugation, and the supernatant was prepared for electrophoresis as described above.
In-vitro translation and immunoprecipitation of WGA. Dry seeds were imbibed overnight in 10-4 M ABA with aeration. Abscisic acid prevents germination of mature wheat embryos and causes continued expression of genes associated with embryogenesis, including WGA (Quatrano et al. 1983). Embryos were excised, frozen under liquid nitrogen, and ground to a powder. Total RNA was isolated using a procedure modified from Jackson and Ingle (1973), Silflow et al. (1979) and Baulcombe and Key (1980). The frozen powder was suspended in extraction buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaC1, 6%, w/v, sodium paminosalicylic acid (Sigma), 1%, w/v, tri-isopropylnaphthalene sulfonic acid (Eastman Kodak, Rochester, N.Y., USA), 6%, v/v, butanol) and extracted with an equal volume of phenolchloroform-isoamyl alcohol (25:24:1, by vol.). The aqueous phase was adjusted to 0.5 M NaC1 and re-extracted twice with the phenol-chloroform-isoamyl alcohol mixture. Nucleic acids were precipitated by adding 0.1 volumes of 3 M Na-acetate and two volumes of cold ethanol. After incubation for 1 h at - 80~ C, the precipitate was pelleted by centrifugation, and redissolved in 10 mM Tris-HC1 (pH 8), 1 mM disodium ethylenediaminetetraacetate (Na2EDTA), 1% (w/v) sodium N-lauroylsarcosine (Sigma), 0.5 M NaC1. After two rounds of phenol
M.A. Mansfield et al.: Processing of wheat-germ agglutinin extraction, nucleic acids in the aqueous phase were precipitated with ethanol as described above. The precipitate was pelleted by centrifugation at 16500-g, washed with 70% ethanol, and redissolved in 10 mM Tris-HC1 (pH 8), 1 mM Na2EDTA, 0.5% (w/v) sodium N-lauroyl sarcosine. Solid NaC1 was added to a concentration of 3 M, and RNA was allowed to precipitate overnight at 4 ~ C. The precipitate was collected by centrifugation, redissolved in 10 mM Tris-HC1 (pH 8), 1 mM Na2EDTA, 0.1% (w/v) SDS. The RNA was precipitated with ethanol once more as described above. Polyadenylated RNA [poly(A)+ RNA] was purified by chromatography on oligo(deoxythymidine)-cellulose (New England Biolabs, Beverly, Mass., USA) as described by Silflow et al. (1979) except that poly(A) +RNA was eluted at room temperature. The amount of poly(A) § RNA was quantified as described in Bishop et al. (1974). Polyadenylated RNA was translated in vitro using a rabbit reticulocyte lysate (Bethesda Research Laboratories, Gaithersburg, Md., USA) supplemented with [35S]methionine (43 000 GBq'mmol- 1; New England Nuclear). Wheat-germ agglutinin was immunoprecipitated from the translation products using a polyclonal serum developed against denatured WGA. All manipulations to obtain the antiserum were as described in Mishkind et al. (1982) except that, prior to injections, WGA was denatured by boiling in 0.01% SDS. The immunoprecipitate was purified with Pansorbin (Calbiochem). Labeled polypeptides were resolved by electrophoresis as described above. Results
Biochemical and immunocytochemical localization of WGA in organelles. T h e p u r p o s e o f t h e first s e t of experiments was to analyze the distribution and c a r b o h y d r a t e - b i n d i n g c a p a c i t y o f W G A in o r g a n elles. D e v e l o p i n g w h e a t e m b r y o s w e r e l a b e l e d w i t h [35S]cysteine t o p r o d u c e h i g h l y l a b e l e d W G A , a n d the soluble and organellar fractions were separated by Sepharose 4B chromatography. Fractions collected from the column were analyzed for WGA content, agglutination activity, and total radioact i v i t y ( F i g . 1). N e w l y s y n t h e s i z e d W G A , i s o l a t e d by N-acetylglucosamine affinity chromatography, w a s f o u n d p r i m a r i l y in t h e o r g a n e l l a r f r a c t i o n ; a s m a l l a m o u n t w a s a l s o d e t e c t e d in t h e s o l u b l e f r a c t i o n . A g g l u t i n a t i o n a c t i v i t y , h o w e v e r , w a s restricted to the soluble fraction, indicating that the b u l k o f t h e u n l a b e l e d W G A e l u t e d w i t h this f r a c tion. Both of these assays for WGA are dependent upon the ability of the protein to bind carbohyd r a t e ; t h i s o c c u r s o n l y w h e n W G A is p r e s e n t in t h e d i m e r i c f o r m ( W r i g h t 1987). D e t e c t i o n o f W G A in t h e o r g a n e l l a r f r a c t i o n w i t h o u t a n a s s o ciated agglutination activity can be attributed to t h e f a c t t h a t a f f i n i t y p u r i f i c a t i o n is m u c h m o r e s e n sitive t h a n t h e a g g l u t i n a t i o n a s s a y . A p o r t i o n o f t h e n e w l y l a b e l e d W G A f o u n d in t h e s o l u b l e f r a c tion may have resulted from disruption of organelles d u r i n g h o m o g e n i z a t i o n ( S t i n i s s e n et al. 1984). Nevertheless, a substantial amount of the newly s y n t h e s i z e d W G A is o r g a n e l l e - a s s o c i a t e d in a d i meric form.
M.A. Mansfield et al. : Processing of wheat-germ agglutinin
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To determine whether WGA is transported out of the organelles, soluble and organellar WGA fractions were isolated from embryos pulse-labeled with [35S]cysteine. Initially, most of the label was found in the organellar fraction (Fig. 2a). As the duration of the pulse was lengthened, the proportion of labeled WGA in the soluble fraction increased. After 1 h of labeling, approx. 70% of the newly synthesized WGA was in the organellar fraction while 30% was in the soluble fraction (Fig. 2 b). When pulse-labeled embryos were transferred to buffer containing unlabeled cysteine, 35Slabeled WGA was chased into the soluble fraction with a half-life of approx. 8 h, indicating that WGA did not remain in the organellar fraction. When nondenatured forms of organellar and soluble WGA were compared by Sephadex G-100 chromatography, organellar WGA eluted more rapidly than soluble WGA (Fig. 3 a) while soluble WGA eluted at a position coincident with mature WGA (Fig. 3 b). Thus, organellar WGA represents pro-WGA and soluble WGA represents mature WGA. The shoulder on the peak of soluble WGA (Fig. 3 b) indicated the presence of a small amount of pro-WGA in the soluble fraction. This may have
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Planta (1988) 173:482-489
9 Springer-Verlag 1988
Wheat-germ agglutinin is synthesized as a glycosylated precursor Michael A. Mansfield 1, Willy J. Peumans 2 and Natasha V. Raikhel i , MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824-1312, USA, and 2 Laboratorium voor Plantenbiochimie, Katholieke Universitat Leuven, Kardinaal Mercierlaan 92, B-3030 Leuven, Belgium
The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [35S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADHcytochrome c reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles. Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with Mr 21000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of Mr 23000 and the mature monomer of Mr 18000. When [aH]mannose was used in labeling studies, only the polypeptide of Mr 23000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of Mr 20000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the Abstract.
* To whom correspondence should be addressed Abbreviations: ER=endoplasmic reticulum; IgG=immunoglobulin G; Mr = relative molecular mass; poly(A) +RNA = polyadenylated RNA; SDS-PAGE=sodium dodecyl sulfatepolyacrylamide gel electrophoresis; WGA = wheat-germ agglutinin
carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745-6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide. K e y w o r d s : Glycoprotein precursor - Lectin - Protein processing - Triticum (wheat germ agglutinin) - Wheat germ agglutinin.
Introduction
At least three classes of vacuolar proteins are found in higher plants: acid hydrolases, lectins and storage proteins. Many of these proteins are synthesized as pre-pro-proteins on the rough endoplasmic reticulum (ER) and undergo cotranslational cleavage of the signal sequence in the lumen of the ER. The polypeptide may also be glycosylated with a glycan which is subject to further modification. In the Golgi apparatus, the polypeptides are packaged into vesicles that deliver them to the vacuoles, where further proteolytic cleavage may take place to yield the mature proteins (for a recent review, see Chrispeels 1984). The patterns of cleavage observed in different plant species are varied. Processing of rice glutelin, pea legumin, pea lectin, and 11S storage proteins of legumes involves conversion of one polypeptide to two subunits (see Higgins 1984 for review). Post-translational modification of a sulfur-rich protein from Brazil nut (Bertholletia excelsa) includes loss of the N-terminal domain (Sun et al. 1987). Loss of several amino acids from the carboxyl-terminal domain has been reported for pea lectin (Higgins et al. 1983) and for napin, a rapeseed storage protein (Crouch et al. 1983; Ericson et al. 1986). In the case of the lectin concanavalin A, posttranslational processing is necessary to generate a functional molecule. The polypeptide sequence un-
M.A. Mansfield et al. : Processing of wheat-germ agglutinin
dergoes several proteolytic cleavages and religation to yield the mature molecule (Bowles et al. 1986; Chrispeels et al. 1986). The biosynthesis of concanavalin A is of particular interest because pro-concanavalin A is glycosylated and the glycan is lost during processing (Herman et al. 1985). When glycosylation of pro-concanavalin A was prevented with tunicamycin, the unglycosylated precursor accumulated in the ER and Golgi (Faye and Chrispeels 1987). This phenomenon, however, may be unique to concanavalin A. Many vacuolar proteins are not glycoproteins (Higgins 1984) and preventing glycosylation of some proteins with tunicamycin did not affect their intracellular accumulation in protein bodies-vacuoles (Chrispeels et al. 1982; Bollini et al. 1983; Lord 1985). Wheat-germ agglutinin (WGA) is a lectin found in the embryos of wheat seeds (Mishkind et al. 1980). The maximum abundance of WGA is several micrograms per embryo (Mishkind et al. 1983), and WGA is retained or synthesized de novo many days after germination (Mishkind et al. 1980; Raikhel et al. 1984a). In wheat embryos, WGA is transported to vacuoles in specific regions of the coleoptile, radicle, coleorhiza, scutellum, and epiblast (Mishkind et al. 1982, 1983). Like many vacuolar proteins, WGA is processed during its transport to the vacuoles. The mature protein is a dimer consisting of two unglycosylated subunits of 171 amino acids each (Wright 1987). Accumulation of WGA in vacuoles implies passage through the ER. Recent analysis of a cDNA for WGA demonstrated the coding potential for a polypeptide extending 15 residues beyond the caroboxy-terminus of the mature protein (Raikhel and Wilkins 1987). This carboxylterminal sequence is more hydrophobic than the rest of the protein and contains a potential glycosylation site. These observations indicate that processing of W G A may be more complex than previously appreciated. In this study we show that pro-WGA is a glycosylated precursor and that the carboxyl-terminal end of the protein and glycan are lost during the transport process. Material and methods Plant material. Wheat (Triticum aestivum L. cv. Marshall; obtained from Minnesota Crop Improvement Association, St. Paul, USA) plants were grown in a growth chamber with a 16-h light period at 25~ and an 8-h dark period at 15~ C. Other growth conditions were as specified in Raikhel and Quatrano (1986). Unless otherwise indicated, experiments were carried out on developing embryos harvested at 20 d post-anthesis. Embryos were squeezed from the grains using forceps and washed free of liquid endosperm in distilled water.
483
Organelle analysis. In-vivo labeling, tissue homogenization, organelle isolation, agglutination assays, and gradient analysis were performed as described in Stinissen et al. (1984). Homogenization buffer A contained 100 mM 2-amino-2-(hydroxymethyl)-l,2-propanediol (Tris)-HCl, pH 7.8, and 12% (w/v) sucrose. Wheat-germ agglutinin in fractions eluted from Sepharose 4B or gradients was affinity-purified on immobilized Nacetylglucosamine (Selectin; Pierce Chemical Co., Rockford, Ill., USA) (Mishkind et al. 1980). Polyaerylamide gel electrophoresis. To optimize the resolution of WGA, proteins were first carboxyamidated (Raikhel et al. 1984a). The polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels (Laemmli 1970). Radioactively labeled proteins were visualized by fluorography with En3Hance (New England Nuclear, Boston, Mass., USA). Tissue fixation and immunoeytochemistry. All procedures, unless otherwise specified, were carried out at room temperature. Immunoreagents were centrifuged at 5000-g for 10 rain prior to use while all other solutions were sterilized by filtration. Embryos were collected and fixed in 3% glutaraldehyde in 25 mM Na-phosphate buffer (pH 7) for 1.5 h at room temperature, post-fixed in 2% OsO4 in the same buffer for 1.5 h, and then washed with additional buffer. Following dehydration in an ethanol series, the tissue was embedded in Spurr's resin (Polysciences, Warrington, Penn., USA). Thin sections were prepared on an Ultracut E microtome (Reichert-Jung, Buffalo, N.Y., USA) and mounted on parlodion-carbon coated gold grids (Polysciences). Immunogold labeling was performed on the grids. Sections were treated with saturated NaIO4 for 10 rain, washed in deionized water, treated with 0.1 N HC1 for 10 rain, and rinsed in deionized water (Craig and Goodchild 1984). Anti-WGA antibody (Mishkind et al. 1982) at a concentration of 30 ~tg.m1-1 in Tris-buffered saline containing 0.1% bovine serum albumin (TBS-BSA: 50raM Tris-HCl, pH 7.8, 150mM NaCI, 0.1% bovine serum albumin (Sigma), 0.05% polyoxyethylenesorbitan monolaurate (Tween-20; Sigma) was then applied for 12 h at 4~ C. After washing in TBS-BSA, the sections were incubated for 1 h with goat-anti-rabbit-immunoglobulinG (IgG) bound to colloidal gold (10 nm; Janssen Pharmaceutica, Beerse, Belgium) diluted 20-fold in TBS-BSA. After immunolabeling, the grids were treated with 1% glutaraldehyde in TBS for 10 rain, washed with deionized water, and then stained with 5% uranyl acetate and lead citrate. Controls included treatment with antiWGA antibody preadsorbed with purified WGA, and treatment with non-immune rabbit-IgG (Sigma) instead of antiWGA antibody. Thin section were examined on a JEOL (Tokyo, Japan) 100CXII transmission microscope. Isolation of WGA from in-vivo labeled embryos. A square of Parafilm (Fisher Scientific, Livonia, Mich., USA) was placed on top of water-saturated Blot Block paper (Schleicher and Schuell, Keene, N.H., USA) in a 100-ram Petri dish. Embryos were placed on the Parafihn in a 400-1xl drop of incubation solution (10-4 M ABA (Sigma) in sterile water). Exogenous ABA enhances synthesis of WGA in isolated embryos (Triplett and Quatrano 1982). After 2 h of preincubation, the solution was removed and replaced with fresh incubation solution containing 1.89 MBq L-[3~S]methionine (43000 GBq.mmol-1; New England Nuclear) or 1.89MBq D-[2,6-3H]mannose (2040 GBq-retool-1; Amersham Corp., Arlington Heights, IlL, USA). All incubations were carried out at room temperature. For the tunicamycin experiments, embryos were cut along the median longitudinal axis with a clean razor blade to facili-
484 tate uptake. The initial incubation solution contained 10 -4 M ABA, 0.5 M NaOH, and 0.5 mg-m1-1 tunicamycin (Sigma). For labeling, the incubation solution contained 10 4 M ABA, 0.5 mg-m1-1 tunicamycin and [35S]methionine as described above. Embryos were homogenized in 600 ~tl of 50 mM sodium acetate (pH 3.8), 0.1 M NaC1, containing 1 mM phenylmethylsulfonyl fluoride (Sigma). The homogenizer was washed with 600 gl of homogenization buffer. The homogenate was centrifuged at 16000.g for 10 min to remove debris, and (NH4)2SO 4 was added to the supernatant to 60% saturation. After 2 h at 4~ C, the precipitate was pelleted. The pelleted was resuspended in buffer B (0.05 M Tris-acetate, pH 5, 0.1 M NaC1) and passed over a N-acetylglucosamine affinity microcolumn (Selectin I; Pierce Chemical Co.) with a bed volume of approx. 75 gl. After washing the column with five 100-gl aliquots of buffer B, bound WGA was eluted with sequential 100 gl and 50-l.tl aliquots of 0.1 M N-acetylglucosamine. The aliquots were combined and lyophilized. The pellet was resuspended in Laemmli sample buffer (Laemmli 1970). For immunoprecipitation, the original supernatant was neutralized with 0.1 volumes of 1 M Tris-HCl (pH 7.8). Nacetylglucosamine was added from a 1 M stock to give a final concentration of 0.1 M. To remove nonspecifically binding proteins, 30 gl of Pansorbin (Calbiochem, San Diego, Cal., USA) cells, pre-washed with immunoprecipitation buffer (20 mM Tris-HC1, pH 7.4, 0.15 M NaC1, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate), were added to the sample. After 25 rain at 4~ C, the Pansorbin cells were removed by centrifugation at 16000.g for 2 rain. The supernatant was then mixed with a rabbit polyclonal serum developed against WGA (Mishkind et al. 1982) and incubated overnight at 4 ~ C. To isolate the WGA-antibody complexes, 50 gl of Pansorbin were added. After incubation at 4 ~ C for 6 h, the Pansorbin cells were pelleted by centrifugation at 16000.g for 2 rain. The supernatant was removed, and the pellet was resuspended in 750 gl of immunoprecipitation buffer. The Pansorbin cells were once again collected by centrifugation. This washing step was repeated three times with immunoprecipitation buffer and once with 50 mM Tris-HC1 (pH 7.4). The final pellet was resuspended in 25 gl of Laemmli sample buffer and heated at 100~ C for 2 rain. The Pansorbin cells were pelleted by centrifugation, and the supernatant was prepared for electrophoresis as described above.
In-vitro translation and immunoprecipitation of WGA. Dry seeds were imbibed overnight in 10-4 M ABA with aeration. Abscisic acid prevents germination of mature wheat embryos and causes continued expression of genes associated with embryogenesis, including WGA (Quatrano et al. 1983). Embryos were excised, frozen under liquid nitrogen, and ground to a powder. Total RNA was isolated using a procedure modified from Jackson and Ingle (1973), Silflow et al. (1979) and Baulcombe and Key (1980). The frozen powder was suspended in extraction buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaC1, 6%, w/v, sodium paminosalicylic acid (Sigma), 1%, w/v, tri-isopropylnaphthalene sulfonic acid (Eastman Kodak, Rochester, N.Y., USA), 6%, v/v, butanol) and extracted with an equal volume of phenolchloroform-isoamyl alcohol (25:24:1, by vol.). The aqueous phase was adjusted to 0.5 M NaC1 and re-extracted twice with the phenol-chloroform-isoamyl alcohol mixture. Nucleic acids were precipitated by adding 0.1 volumes of 3 M Na-acetate and two volumes of cold ethanol. After incubation for 1 h at - 80~ C, the precipitate was pelleted by centrifugation, and redissolved in 10 mM Tris-HC1 (pH 8), 1 mM disodium ethylenediaminetetraacetate (Na2EDTA), 1% (w/v) sodium N-lauroylsarcosine (Sigma), 0.5 M NaC1. After two rounds of phenol
M.A. Mansfield et al.: Processing of wheat-germ agglutinin extraction, nucleic acids in the aqueous phase were precipitated with ethanol as described above. The precipitate was pelleted by centrifugation at 16500-g, washed with 70% ethanol, and redissolved in 10 mM Tris-HC1 (pH 8), 1 mM Na2EDTA, 0.5% (w/v) sodium N-lauroyl sarcosine. Solid NaC1 was added to a concentration of 3 M, and RNA was allowed to precipitate overnight at 4 ~ C. The precipitate was collected by centrifugation, redissolved in 10 mM Tris-HC1 (pH 8), 1 mM Na2EDTA, 0.1% (w/v) SDS. The RNA was precipitated with ethanol once more as described above. Polyadenylated RNA [poly(A)+ RNA] was purified by chromatography on oligo(deoxythymidine)-cellulose (New England Biolabs, Beverly, Mass., USA) as described by Silflow et al. (1979) except that poly(A) +RNA was eluted at room temperature. The amount of poly(A) § RNA was quantified as described in Bishop et al. (1974). Polyadenylated RNA was translated in vitro using a rabbit reticulocyte lysate (Bethesda Research Laboratories, Gaithersburg, Md., USA) supplemented with [35S]methionine (43 000 GBq'mmol- 1; New England Nuclear). Wheat-germ agglutinin was immunoprecipitated from the translation products using a polyclonal serum developed against denatured WGA. All manipulations to obtain the antiserum were as described in Mishkind et al. (1982) except that, prior to injections, WGA was denatured by boiling in 0.01% SDS. The immunoprecipitate was purified with Pansorbin (Calbiochem). Labeled polypeptides were resolved by electrophoresis as described above. Results
Biochemical and immunocytochemical localization of WGA in organelles. T h e p u r p o s e o f t h e first s e t of experiments was to analyze the distribution and c a r b o h y d r a t e - b i n d i n g c a p a c i t y o f W G A in o r g a n elles. D e v e l o p i n g w h e a t e m b r y o s w e r e l a b e l e d w i t h [35S]cysteine t o p r o d u c e h i g h l y l a b e l e d W G A , a n d the soluble and organellar fractions were separated by Sepharose 4B chromatography. Fractions collected from the column were analyzed for WGA content, agglutination activity, and total radioact i v i t y ( F i g . 1). N e w l y s y n t h e s i z e d W G A , i s o l a t e d by N-acetylglucosamine affinity chromatography, w a s f o u n d p r i m a r i l y in t h e o r g a n e l l a r f r a c t i o n ; a s m a l l a m o u n t w a s a l s o d e t e c t e d in t h e s o l u b l e f r a c t i o n . A g g l u t i n a t i o n a c t i v i t y , h o w e v e r , w a s restricted to the soluble fraction, indicating that the b u l k o f t h e u n l a b e l e d W G A e l u t e d w i t h this f r a c tion. Both of these assays for WGA are dependent upon the ability of the protein to bind carbohyd r a t e ; t h i s o c c u r s o n l y w h e n W G A is p r e s e n t in t h e d i m e r i c f o r m ( W r i g h t 1987). D e t e c t i o n o f W G A in t h e o r g a n e l l a r f r a c t i o n w i t h o u t a n a s s o ciated agglutination activity can be attributed to t h e f a c t t h a t a f f i n i t y p u r i f i c a t i o n is m u c h m o r e s e n sitive t h a n t h e a g g l u t i n a t i o n a s s a y . A p o r t i o n o f t h e n e w l y l a b e l e d W G A f o u n d in t h e s o l u b l e f r a c tion may have resulted from disruption of organelles d u r i n g h o m o g e n i z a t i o n ( S t i n i s s e n et al. 1984). Nevertheless, a substantial amount of the newly s y n t h e s i z e d W G A is o r g a n e l l e - a s s o c i a t e d in a d i meric form.
M.A. Mansfield et al. : Processing of wheat-germ agglutinin
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Fig. 1. Fractionation of extracts from pulse-labeled wheat embryos on Sepharose 4B. Embryos (150) were pulse-labeled with 750 kBq [3SS]cysteine, homogenized in buffer A, and fractionated on Sepharose 4B. Fractions (1 ml) were collected, and 10-gl aliquots were assayed for agglutination activity ( a - - a ) , expressed as the titer (highest dilution at which agglutination still occurs) of the fraction, and for measurement of [35S]cysteine incorporation into TCA-insoluble material ( o - - o ) , expressed as cpm. Triton X-~00 was added to the remainder of each fraction to a final concentration of 0.5%. The WGA was isolated using N-acetylglucosamine affinity chromatography and the radioactivity in WGA, expressed as cpm, was determined ( e - - - e )
To determine whether WGA is transported out of the organelles, soluble and organellar WGA fractions were isolated from embryos pulse-labeled with [35S]cysteine. Initially, most of the label was found in the organellar fraction (Fig. 2a). As the duration of the pulse was lengthened, the proportion of labeled WGA in the soluble fraction increased. After 1 h of labeling, approx. 70% of the newly synthesized WGA was in the organellar fraction while 30% was in the soluble fraction (Fig. 2 b). When pulse-labeled embryos were transferred to buffer containing unlabeled cysteine, 35Slabeled WGA was chased into the soluble fraction with a half-life of approx. 8 h, indicating that WGA did not remain in the organellar fraction. When nondenatured forms of organellar and soluble WGA were compared by Sephadex G-100 chromatography, organellar WGA eluted more rapidly than soluble WGA (Fig. 3 a) while soluble WGA eluted at a position coincident with mature WGA (Fig. 3 b). Thus, organellar WGA represents pro-WGA and soluble WGA represents mature WGA. The shoulder on the peak of soluble WGA (Fig. 3 b) indicated the presence of a small amount of pro-WGA in the soluble fraction. This may have
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