Plant Plasma Membrane Proteins1

Plant Physiol. (1987) 85, 1048-1054 0032-0889/87/85/ 1048/00/$0 1.00/0

Plant Plasma Membrane Proteins1 IMMUNOLOGICAL CHARACTERIZATION OF A MAJOR 75 KILODALTON PROTEIN GROUP Received for publication May 6, 1987 and in revised form August 10, 1987

HOWARD D. GRIMES*2 AND R. WILLIAM BREIDENBACH

Plant Growth Laboratory, University ofCalifornia-Davis, Davis, California 95616 tial problem with monoclonal antibodies raised against plasma membrane proteins and that is an apparent lack of specificity. A major 75 kD protein group from the tomato plasma membrane was By definition, a monoclonal antibody is specific to a single semipurified on polyacrylamide gels and used to raise a rabbit antiserum. epitope (18). This single epitope, however, may be found on The resulting antiserum recognized a single 75 kilodalton band from more than one protein in a given membrane system. For inphase partitioned tomato plasma membrane (from both sispension cells stance, if a monoclonal antibody was generated against a manand mature, green fruit) after resolution on one-dimensional polyacryl- nose-mannose dimer in a glycosyl chain, then there is a high amide gels. Two-dimensional polyacrylamide gel analysis of proteins probability of finding that epitope on other proteins. This probfrom tomato plasma membrane showed that the 75 kilodalton antiserum lem may be exacerbated when membrane preparations are used recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons to generate the antibodies, because of the high degree of glyco(mean = 75.6 kilodaltons) and with isoelectric point values ranging from sylation associated with membrane proteins, especially those of 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This the plasma membrane (9). It was shown that monoclonal antiantiserum was shown to bind protoplast surface epitopes by indirect bodies raised against purified brain plasma membrane recognized immunofluorescence. The presence of this protein group in both mono- many proteins when immunoblotting assays were performed cotyledonous and dicotyledonous plants was established by immunoblot- against proteins of the brain plasma membrane (6). ting the tomato 75 kilodalton antiserum against proteins obtained from The probability of generating antibodies against carbohydrate plasma membrane-enriched fractions from corn roots and soybean roots. epitopes may be even higher in plant membranes for two reasons. The data suggest that this 75 kilodalton protein group is a major First, cell wall fragments may co-purify with plasma membrane proteinaceous component of the plant plasma membrane. resulting in the presentation of large amounts of carbohydrate antigen to the animal immune system. Second, the plant plasma membrane appears to be heavily glycosylated, approximately 20% by weight (HD Grimes, unpublished data), compared to animal plasma membrane (0.3-3% by weight; 15). Obviously, antibodies that recognize carbohydrate epitopes have little potential as probes for molecular genetic studies. Proteins are abundant constituents of the plant plasma memFor the reasons outlined above, we chose to generate polybrane, an organelle which mediates many vital cellular functions, clonal antibodies against purified tomato plasma membrane including ion transport (27), cell wall synthesis and assembly antigens. Since polyclonal antibodies recognize more than one (34), and the response to hormonal and environmental signals epitope on a given protein, they could prove to be more effective (1). Although there are as many as 100 polypeptides visible on as probes for molecular genetic studies. In the present report, we two-dimensional gels of plasma membrane preparations from describe the production of a polyclonal antiserum to a group of plant cells (4), only one of these proteins, the H+-ATPase, has a 75 kD proteins. known function (5, 12, 25, 27, 30) and can be located after SDSPAGE. Since no functional assays exist for any other proteins of MATERIALS AND METHODS the plant plasma membrane, it is imperative that other probes be developed that will allow identification of specific proteins. Tomato Cells. Tomato cell line No. 741505-45, an interspecific Antibodies, either polyclonal or monoclonal, could provide an hybrid of Lycopersicon esculentum and L. peruvianum, was ideal solution to this problem. Immunological probes to plasma obtained from D. Pratt (University of California, Davis). This membrane proteins would facilitate identification, purification, was kept as a suspension culture on a Linsmaier and Skoog and characterization of these proteins as well as providing pow- based medium (23) with 2 mg/L 2,4-dichlorophenoxyacetic acid erful tools for the molecular analysis of plasma membrane pro- and 1 mg/L 2-isopentenyl adenine. A 1:10 dilution was pertein synthesis and processing. formed when the culture was in late log phase (usually 6 d after Antibodies, both polyclonal and monoclonal, against plasma the previous dilution) and the cells were harvested for experimembrane proteins have already proven extremely valuable in ments in mid-log phase or 4 d after dilution. many animal systems (3, 14, 16, 17, 29, 32). Norman et al. (24) Tomato Fruit. Mature, green tomato fruit were obtained from and Villanueva et al. (32) have recently described the production a field planting of L. esculentum (cv Castlemart). of several monoclonal antibodies to proteins from tobacco and Protoplast Isolation from Suspension Cultured Cells. Cells soybean membranes, respectively. There is, however, one poten- were collected by filtration on a Buchner funnel with Whatman No. I filter paper and washed with 10 to 20 ml of protoplast 'The authors wish to thank Beatrice Foods and the Department of digestion buffer (400 mM mannitol, 4.3 g/L Murashige and Skoog salts (Gibco), 3 mM Mes, 7 mM CaNO3,0.1 % BSA, 0.1 S% gelatin). Energy for supporting portions of this research. 2 Present address: Department of Botany and Plant Pathology, Lilly Approximately 20 g fresh weight cells were suspended in 100 ml protoplast digestion buffer containing 1200 units pectinase and Hall of Life Sciences, Purdue University, West Lafayette, IN 47907. ABSTRACT

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PLANT PLASMA MEMBRANE PROTEINS

Table I. Marker Enzyme Assessment of Tomato Fruit-Derived Plasma Membrane Purity after Aqueous Two-Phase Partitioning Lower Upper Phase Phase Vanadate-sensitive ATPasea 2.22 9.91 17.8 ,umol/h 1.94 8.24 2.46 jumol/h/mg protein Latent IDPase 27.6 38.4 0.192 rmol/h 8.2 0.16 6.4 gmol/h/mg protein Cyt c oxidase 16.1 0.024 18.4 ,umol/min 6.3 0.009 2.57 jsmol/min/mg protein

10,000 units cellulase (Cooper Biomedicals). After approximately 3 h, the protoplast suspension was filtered through four layers of cheesecloth and then centrifuged at 40g for 5 min. The protoplast pellet was washed twice by gentle suspension in protoplast digestion buffer. Tissue Homogenization. Isolated protoplasts were diluted (1:3 packed cell volume to buffer) with ice-cold tomato homogenization buffer (0.46 M sucrose, 3 mM EDTA, 3 mM dithiothreitol, 25 mm Tris/Mes [pH 7.2]) and 0.5% polyvinylpolypyrrolidone. All subsequent procedures were carried out on ice or in a cold room. Protoplasts were homogenized with a polytron for 10 s at low speed. The brie was centrifuged at 4,300g for 12 min. The supernatant was centrifuged at 120,000g for 30 min. The resulting crude membrane pellet was resuspended in 250 mm sucrose, 5 mm potassium phosphate (pH 7.8) to a concentration of about 8 mg/ml. Tomato fruit was quartered and all locular tissue was removed leaving only the pericarp and epidermal tissue. Each IOOg of tissue was homogenized with 200 ml of tomato fruit homogenization buffer (250 mm sucrose, 70 mM Tris, 10 mM BisTrisPropane, 3 mm DTT, 0.1% BSA, 0.5% PVP by blending for 1 min at high speed in a Waring Blendor. The brei was filtered through four layers of cheesecloth and centrifuged at 10,000g for 15 min. Supematant was centrifuged at 120,000g for 30 min. The resulting crude membrane pellet was resuspended in 250 mm sucrose, 5 mm potassium phosphate (pH 7.8) to a concentration of about 15 mg/ml. Aqueous Two-Phase Partitioning of Plasma Membrane. A 2 ml sample of the resuspended crude membrane pellet was added to a premade, 8.0g, two-phase system. The two-phase system was prepared according to Hodges and Mills (13) and consisted of 6.5% (w/w) Dextran T500 (Pharmacia), 6.5% polyethylene gly-

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UDP-['4-C]-galactose: galactosyltransferase 1120 NDb 12,165 cpm/mg protein 0.212 ND 0.019 nmol/0.5h/mg protein a Assayed in the presence of 0.02% Triton X-100 and 50 mm b Not determined. KC1.

col 3350 (Sigma), 0.125 M sucrose, 0.5 mM KCI, and 3.3 mM potassium phosphate (pH 7.8). Final weight of the two-phase mixture was brought to 8.0 g with cold deionized H20. The twophase system was then vigorously mixed by inverting the tube 20 times. Phase separation was accelerated by centrifugation at 2400g for 10 min. For suspension cells, the first upper phase was collected, diluted 10-fold with tomato homogenization buffer, and centrifuged at 120,000g for 30 min. For tomato fruit, the upper phase was collected and washed

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peptide band of 75 kD. A, Coomassie brilliant blue stain of tomato membrane fractions. 1, Mol wt markers; 2, plasma membrane isolated from tomato fruit; 3, plasma membrane isolated from tomato protoplasts; 4, lower phase from tomato protoplasts; 100 sg protein was loaded per lane. B, Immunoblot with 75 kD antiserum against tomato membrane fractions. 1, Plasma membrane isolated from tomato fruit; 2, plasma membrane isolated from tomato protoplasts; 3, lower phase from tomato protoplasts. Antiserum dilution was 1:500; 60 Mg protein was loaded per lane. Preimmune serum did not stain any bands.

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Plant Physiol. Vol. 71, 1987

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