plant plasma membrane proteins

Plant Physiol. (1987) 85, 1048-10540032-0889/87/85/ 1048/00/$0 1.00/0

Plant Plasma Membrane Proteins1IMMUNOLOGICAL 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 BREIDENBACHPlant Growth Laboratory, University ofCalifornia-Davis, Davis, California 95616

ABSTRACT

A major 75 kD protein group from the tomato plasma membrane wassemipurified on polyacrylamide gels and used to raise a rabbit antiserum.The resulting antiserum recognized a single 75 kilodalton band fromphase partitioned tomato plasma membrane (from both sispension cellsand mature, green fruit) after resolution on one-dimensional polyacryl-amide gels. Two-dimensional polyacrylamide gel analysis of proteinsfrom tomato plasma membrane showed that the 75 kilodalton antiserumrecognized a group of proteins ranging from 63.1 to 88.2 kilodaltons(mean = 75.6 kilodaltons) and with isoelectric point values ranging from5.7 to 6.3. No other spots were visible on the two-dimensional blots. Thisantiserum was shown to bind protoplast surface epitopes by indirectimmunofluorescence. The presence of this protein group in both mono-cotyledonous and dicotyledonous plants was established by immunoblot-ting the tomato 75 kilodalton antiserum against proteins obtained fromplasma membrane-enriched fractions from corn roots and soybean roots.The data suggest that this 75 kilodalton protein group is a majorproteinaceous component of the plant plasma membrane.

Proteins are abundant constituents of the plant plasma mem-brane, an organelle which mediates many vital cellular functions,including ion transport (27), cell wall synthesis and assembly(34), and the response to hormonal and environmental signals(1). Although there are as many as 100 polypeptides visible ontwo-dimensional gels of plasma membrane preparations fromplant cells (4), only one of these proteins, the H+-ATPase, has aknown function (5, 12, 25, 27, 30) and can be located after SDS-PAGE. Since no functional assays exist for any other proteins ofthe plant plasma membrane, it is imperative that other probesbe developed that will allow identification of specific proteins.Antibodies, either polyclonal or monoclonal, could provide anideal solution to this problem. Immunological probes to plasmamembrane proteins would facilitate identification, purification,and characterization of these proteins as well as providing pow-erful tools for the molecular analysis of plasma membrane pro-tein synthesis and processing.

Antibodies, both polyclonal and monoclonal, against plasmamembrane proteins have already proven extremely valuable inmany animal systems (3, 14, 16, 17, 29, 32). Norman et al. (24)and Villanueva et al. (32) have recently described the productionof several monoclonal antibodies to proteins from tobacco andsoybean membranes, respectively. There is, however, one poten-

'The authors wish to thank Beatrice Foods and the Department ofEnergy for supporting portions of this research.

2 Present address: Department of Botany and Plant Pathology, LillyHall of Life Sciences, Purdue University, West Lafayette, IN 47907.

tial problem with monoclonal antibodies raised against plasmamembrane proteins and that is an apparent lack of specificity.By definition, a monoclonal antibody is specific to a singleepitope (18). This single epitope, however, may be found onmore than one protein in a given membrane system. For in-stance, if a monoclonal antibody was generated against a man-nose-mannose dimer in a glycosyl chain, then there is a highprobability of finding that epitope on other proteins. This prob-lem may be exacerbated when membrane preparations are usedto generate the antibodies, because of the high degree of glyco-sylation associated with membrane proteins, especially those ofthe plasma membrane (9). It was shown that monoclonal anti-bodies raised against purified brain plasma membrane recognizedmany proteins when immunoblotting assays were performedagainst proteins of the brain plasma membrane (6).The probability of generating antibodies against carbohydrate

epitopes may be even higher in plant membranes for two reasons.First, cell wall fragments may co-purify with plasma membraneresulting in the presentation of large amounts of carbohydrateantigen to the animal immune system. Second, the plant plasmamembrane appears to be heavily glycosylated, approximately20% by weight (HD Grimes, unpublished data), compared toanimal plasma membrane (0.3-3% by weight; 15). Obviously,antibodies that recognize carbohydrate epitopes have little poten-tial as probes for molecular genetic studies.

For the reasons outlined above, we chose to generate poly-clonal antibodies against purified tomato plasma membraneantigens. Since polyclonal antibodies recognize more than oneepitope on a given protein, they could prove to be more effectiveas probes for molecular genetic studies. In the present report, wedescribe the production of a polyclonal antiserum to a group of75 kD proteins.

MATERIALS AND METHODS

Tomato Cells. Tomato cell line No. 741505-45, an interspecifichybrid of Lycopersicon esculentum and L. peruvianum, wasobtained from D. Pratt (University of California, Davis). Thiswas kept as a suspension culture on a Linsmaier and Skoogbased medium (23) with 2 mg/L 2,4-dichlorophenoxyacetic acidand 1 mg/L 2-isopentenyl adenine. A 1:10 dilution was per-formed when the culture was in late log phase (usually 6 d afterthe previous dilution) and the cells were harvested for experi-ments in mid-log phase or 4 d after dilution.Tomato Fruit. Mature, green tomato fruit were obtained from

a field planting of L. esculentum (cv Castlemart).Protoplast Isolation from Suspension Cultured Cells. Cells

were collected by filtration on a Buchner funnel with WhatmanNo. I filter paper and washed with 10 to 20 ml of protoplastdigestion buffer (400 mM mannitol, 4.3 g/L Murashige and Skoogsalts (Gibco), 3 mM Mes, 7 mM CaNO3,0.1% BSA, 0.1S% gelatin).Approximately 20 g fresh weight cells were suspended in 100 mlprotoplast digestion buffer containing 1200 units pectinase and

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PLANT PLASMA MEMBRANE PROTEINS

10,000 units cellulase (Cooper Biomedicals). After approximately3 h, the protoplast suspension was filtered through four layers ofcheesecloth and then centrifuged at 40g for 5 min. The protoplastpellet was washed twice by gentle suspension in protoplast diges-tion buffer.

Tissue Homogenization. Isolated protoplasts were diluted (1:3packed cell volume to buffer) with ice-cold tomato homogeni-zation 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 coldroom. Protoplasts were homogenized with a polytron for 10 s atlow speed. The brie was centrifuged at 4,300g for 12 min. Thesupernatant was centrifuged at 120,000g for 30 min. The result-ing crude membrane pellet was resuspended in 250 mm sucrose,5 mm potassium phosphate (pH 7.8) to a concentration of about8 mg/ml.Tomato fruit was quartered and all locular tissue was removed

leaving only the pericarp and epidermal tissue. Each IOOg oftissue was homogenized with 200 ml of tomato fruit homogeni-zation buffer (250 mm sucrose, 70 mM Tris, 10 mM BisTris-Propane, 3 mm DTT, 0.1% BSA, 0.5% PVP by blending for 1min at high speed in a Waring Blendor. The brei was filteredthrough four layers of cheesecloth and centrifuged at 10,000g for15 min. Supematant was centrifuged at 120,000g for 30 min.The resulting crude membrane pellet was resuspended in 250mm sucrose, 5 mm potassium phosphate (pH 7.8) to a concen-tration of about 15 mg/ml.Aqueous Two-Phase Partitioning of Plasma Membrane. A 2

ml sample of the resuspended crude membrane pellet was addedto a premade, 8.0g, two-phase system. The two-phase system wasprepared according to Hodges and Mills (13) and consisted of6.5% (w/w) Dextran T500 (Pharmacia), 6.5% polyethylene gly-

1 2 34 1

200 I116-92.5 -

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Table I. Marker Enzyme Assessment ofTomato Fruit-Derived PlasmaMembrane Purity after Aqueous Two-Phase Partitioning

Upper LowerPhase Phase

Vanadate-sensitiveATPasea,umol/h 17.8 9.91 2.22jumol/h/mg protein 2.46 8.24 1.94

Latent IDPasermol/h 38.4 0.192 27.6gmol/h/mg protein 6.4 0.16 8.2

Cyt c oxidase,umol/min 18.4 0.024 16.1jsmol/min/mg protein 2.57 0.009 6.3

UDP-['4-C]-galactose:galactosyltransferase

cpm/mg protein NDb 1120 12,165nmol/0.5h/mg protein ND 0.019 0.212a Assayed in the presence

KC1. b Not determined.of 0.02% Triton X-100 and 50 mm

col 3350 (Sigma), 0.125 M sucrose, 0.5 mM KCI, and 3.3 mMpotassium phosphate (pH 7.8). Final weight of the two-phasemixture was brought to 8.0 g with cold deionized H20. The two-phase system was then vigorously mixed by inverting the tube20 times. Phase separation was accelerated by centrifugation at2400g for 10 min.

For suspension cells, the first upper phase was collected, diluted10-fold with tomato homogenization buffer, and centrifuged at120,000g for 30 min.For tomato fruit, the upper phase was collected and washed

3

_-130FIG. 1. 75 kD Antiserum recognizes a

- 75 peptide band of 75 kD. A, Coomassie bril-liant blue stain of tomato membrane frac-

_ 50 tions. 1, Mol wt markers; 2, plasma mem-brane isolated from tomato fruit; 3, plasmamembrane isolated from tomato proto-plasts; 4, lower phase from tomato proto-

- 39 plasts; 100 sg protein was loaded per lane.B, Immunoblot with 75 kD antiserumagainst tomato membrane fractions. 1,

-27 Plasma membrane isolated from tomatofruit; 2, plasma membrane isolated fromtomato protoplasts; 3, lower phase fromtomato protoplasts. Antiserum dilution was

1 7 1:500; 60 Mg protein was loaded per lane.Preimmune serum did not stain any bands.

AB

1049

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GRIMES AND BREIDENBACH

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FIG. 2. 75 kD Antiserum recognizes a major protein group resolved on two-dimensional gels. A, Amido black stain of proteins from the plasmamembrane fraction of tomato fruit after two-dimensional gel electrophoresis and transfer to nitrocellulose; 175 /Ag protein was loaded on the gel. B,Immunoblot of 75 kD antiserum against proteins from the plasma membrane fraction of tomato fruit (175 Ag membrane protein, 1:500 dilution ofthe 75 kD antiserum).

by vigorously mixing with a fresh lower phase. The lower phasewas reextracted by mixing with a fresh upper phase. After phaseseparation, the upper phase from the reextraction was vigorouslymixed with a fresh lower phase and phase separated. It wasnecessary to wash the upper phase with a fresh lower phase dueto the high number of chloroplasts in green, mature tomato fruit.The final upper phases were diluted 10-fold with tomato homog-enization buffer and centrifuged at 120,000g for 30 min. Theplasma membrane pellets were resuspended in 0.46 M sucrose; 5mM Mes (pH 7.5) and either used for experiments or frozen at-80°C and kept for up to 4 weeks.Enzyme Assays. Protein was measured using a modification

of the Bradford method (10) in which 60 ,d of 0.2% Triton X-100 detergent was included in each assay tube to solubilizemembrane proteins. Cyt c oxidase and antimycin A-insensitiveNADH-Cyt c reductase were assayed according to Hodges andLeonard (12). H+-ATPase activity was measured according toHodges and Leonard (12) except that 0.1 mm molybdate and 1mm sodium azide were included in the reaction mixture. LatentIDPase activity was measured in the presence of 0.05% TritonX- 100. Other parameters were identical to Hodges and Leonard(12). UDP-galactose:diacylglycerol galactosyltransferase wasmeasured by the method described by Douce et al. (7).

Electrophoresis. Membrane proteins were resuspended in aLaemmli sample buffer (20) supplemented with 2.5 M urea.Insoluble material was removed by centrifugation prior to gelloading. Polyacrylamide gels were run according to Laemmli(20), except for the addition of 1.0 M urea and that a 10 to 15%acrylamide gradient was used with an accompanying 10 to 15%glycerol gradient. Transfer of proteins to nitrocellulose was per-formed as described by Towbin et al. (3 1).

Antibody Production. Preparative electrophoresis of 700 ,ug ofplasma membrane protein from suspension cultures was resolvedto obtain the antigen. Three narrow strips from the middle andboth edges of the gel were excised and stained with Coomassiebrilliant blue. The 75 kD protein(s) was identified and recovered

from the unstained portion of the gel. The gel was cut into smallpieces, mixed with phosphate buffered saline, and homogenizedto a uniform consistency with a glass/plastic Dounce-type ho-mogenizer. A total volume of 750 ul was recovered. A 150 1,usample was emulsified 1:1 with Freunds complete adjuvant andinjected intradermally into a rabbit. Booster injections consistedof 150 ,ul aliquots with no additives and were given every week.The rabbit was bled from the ear vein 10 d after the third boosterto determine the titer and specificity ofthe antiserum. The rabbitwas terminally bled by cardiac puncture 7 d after the final boosterinjection. The serum obtained is referred to as the 75 kD anti-serum in this paper.

Affinoblotting. Affinoblotting (for carbohydrate visualization)was carried out essentially as described by Faye and Chrispeels(8). After transfer of protein to nitrocellulose, the sheets werefixed with acetic acid:isopropyl alcohol:H20 (10:25:65, v/v),rinsed several times with deionized H20, then with Tris-bufferedsaline (500 mm NaCl; 20 mM Tris [pH 7.5]). The nitrocellulosesheet was incubated for at least 60 min in blocking solution(Tris-buffered saline + 3% gelatin). The blot was then incubatedin 25 ,ug/ml concanavalin A (Sigma), washed in Tris-bufferedsaline + 0.1% Tween 20 (Bio-Rad), and then incubated in 50,ug/ml horseradish peroxidase (Sigma) followed by washing.Color was developed by immersing the blot in 60 mg 4-chloro-l-napthol (Bio-Rad) and 60 ,d ofH202 in 100 ml ofTris-bufferedsaline.

Hydrolysis of Oligosaccharide Chains on NitrocelluloseSheets. Tomato fruit plasma membrane proteins (75 ,ug/lane)were resolved on one-dimensional gels. After transferring tonitrocellulose sheets, the protein was fixed and blocked as de-scribed by Faye and Chrispeels (8). The nitrocellulose strips wereincubated in 5 ml of 50 mm sodium citrate (pH 6.0) containing60 munits of endo H (Genzyme) for 48 h at 37°C. After thisincubation, the strips were washed for 24 h in 10 changes of Tris-buffered saline + 0.1% Tween-20. This was found to be essentialfor subsequent immunoblotting. Decreasing the time of washing

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FIG. 3. Deglycosylation of proteins from tomato fruit plasma mem-brane does not impair binding of the 75 kD antiserum to the 75 kDprotein group. Lanes 1 and 2 are stained with ConA (l is a buffer controland 2 was treated with endo H). Lanes 3 and 4 were stained with the 75kD antiserum (3 was a buffer control and 4 was treated with endo H);60 jg protein per lane. The arrow opposite lane I indicates where thebottom of the 75 kD protein should run.

resulted in a dramatic loss of sensitivity in the immunoblottingassay (data not shown).

Immunofluorescence Visualization of Antibody Binding to theProtoplast Surface. Protoplasts were prepared as described aboveand washed in 400 mM mannitol, 10 mM Hepes (pH 7.0). Isolatedprotoplasts were incubated for 5 min in 400 mm mannitol, 10mM Hepes (pH 7.0) supplemented with goat IgG fraction (1 mg/ml, Behring Diagnostics). This reduced nonspecific binding ofthe secondary antibody. Protoplasts were incubated for 45 minin the 75 kD antibody (mixed with dry mannitol to a finalosmotic concentration of 400 mM). The protoplasts were washedtwo times by centrifugation and resuspension in 400 mm man-nitol, 10 mm Hepes (pH 7.0), and then incubated for 45 min in400 mM mannitol, 10 mM Hepes (pH 7.0), 1 mg/ml BSA, 0.1mg/ml goat IgG (pH 7.0) containing rhodamine-conjugated goat-anti-rabbit antibody (diluted 1:10 in same buffer, Tago Immu-nologicals). Protoplasts were washed three times by centrifuga-tion and resuspension in 400 mM mannitol, 10 mm Hepes (pH7.0). Immunofluorescence was observed with a Zeiss microscope.The excitation wavelength was 546 nm and emission was 590nm.

RESULTSIsolation of Plasma Membrane. Two sources of plasma mem-

brane were used for this investigation; protoplasts isolated from

FIG. 4. Indirect immunofluorescence demonstrates that the 75 kDantiserum labels the protoplast surface- and 2 are stained with 75 kDantiserum, 3 and 4 are stained with preimmune serum. Fluorescentimages are shown in 2 and 4. See text for experimental details.

tomato suspension cells and mature green tomato fruit. We havepreviously reported on the purity of plasma membrane obtainedfrom protoplasts derived from suspension cells (11). The samemethods proved effective for mature green tomato fruit. Table Ipresents data on the purity of plasma membrane from tomatofruit obtained by aqueous two-phase partitioning. The K+-stim-ulated, vanadate-sensitive, H+-ATPase is used most commonlyas the plant plasma membrane marker (12, 30). Approximately50% of the total activity of this enzyme is recovered in the upperphase. There is a 3.2-fold increase in the specific activity foundin the upper phase when compared to the microsome (=crudemembrane pellet) activity. Furthermore, K+ stimulated this ac-tivity by 22%, and the inclusion of 0.02% Triton X-100 wasnecessary to detect any activity. This latter observation indicatesthat the plasma membrane vesicles were sealed and right-side-out(ll, 13, 19,22).The activities ofCyt c oxidase and latent IDPase incorporation

were diminished in the upper phase from tomato fruit. UDP-galactose:diacylglycerol galactosyltransferase activity was parti-tioned predominantly into the lower phase (Table I). This showedthat there was very little contamination of the upper phaseplasma membrane with membrane fragments from the mito-chondria, Golgi apparatus, and chloroplast envelope, respec-tively.

Production and Initial Characterization of the Anti-75 kDAntiserum. Two criteria were used to select which protein(s)would be used to raise antibodies. First, the protein should behighly enriched in the plasma membrane fraction. Second, theprotein should not bind ConA.3 Preliminary one-dimensionalPAGE analyses of proteins from the plasma membrane of sus-pension cells indicated that a prominant protein band of 75 kDmet both of these criteria. Figure IA shows that the 75 kDprotein band was a major component of the plasma membranefrom both tomato protoplasts and fruit. This protein did notbind ConA (Fig. 3). The 75 kD protein band was therefore

'Abbreviation: ConA, concanavalin A.

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A BFIG. 5. 75 kD antiserum recognizes epitopes present in membrane fractions obtained from corn and soybean root. A, Coomassie stained proteins

from 1, mol wt markers; 2, plasma membrane from tomato fruit; 3, plasma membrane-enriched fraction from corn root; and 4, plasma membrane-enriched fraction from soybean root; 100 Mg protein was loaded per lane. B, Immunoblot of 1, plasma membrane from tomato fruit; 2, plasmamembrane-enriched fraction from corn root; and 3, plasma membrane-enriched fraction from soybean root against 75 kD antiserum (1:250 dilution);75 ug protein was loaded per lane.

chosen as a primary target for antibody production.Preparative gel electrophoresis was used to purify the 75 kD

protein band from plasma membranes of suspension cells. Thisregion of the gel was excised and used to raise antiserum. FigurelB shows the results of an immunoblot assay against proteinsfrom the plasma membrane of suspension cells, proteins fromthe plasma membrane of tomato fruit, and proteins from thelower phase ofsuspension cells. The latter includes the tonoplast,endoplasmic reticulum, mitochondria, and chloroplasts and/orplastids, and Golgi apparatus. It was evident that the 75 kDantiserum recognized the protein in both suspension cell andtomato fruit plasma membrane. This antiserum detected a pro-tein in the lower phase membranes, but the staining was barelyvisible (Fig. 1B). This result further indicated that the 75 kDprotein was plasma membrane associated. Preimmune serumdid not detect any bands on immunoblots (data not shown).Two-dimensional gel electrophoresis of proteins from the

plasma membrane fraction of tomato fruit was used to furthercharacterize the antigenic determinants of the 75 kD antiserum.It was evident from Figure 2 that the 75 kD antiserum recognizeda major group of proteins. This major group of proteins has amean M, of 75,600 with a range of 63,100 to 88, 200. The meanisoelectric point of this protein group is 5.9 with a range of 5.7to 6.3.

Although the 75 kD protein group did not appear to bind ConA (Fig. 3; lane 1), it was of interest to determine whether the 75

kD antiserum recognized peptide epitopes. To investigate thispossibility, the proteins of the plasma membrane from tomatofruit were resolved on one-dimensional gels, transferred to nitro-cellulose, and treated with endo H. This glycosidase cleaves inthe core region of the glycosyl group. Figure 3 demonstrates thatendo H treatment effectively removed most ofthe ConA bindingassociated with plasma membrane proteins. Since endo H treat-ment did not impair the 75 kD antiserum binding to the 75 kDprotein group, the 75 kD antiserum probably recognizes peptideepitopes ofthe 75 kD protein group. Because all types ofglycosylchains do not bind ConA, a second experiment was done. Afterthe plasma membrane was isolated from protoplasts, the proteinswere precipitated in 80% acetone and chemically deglycosylatedwith trifluoroacetic acid. After this treatment, the 75 kD anti-serum strongly reacted with the 75 kD epitope (data not shown).Tomato Protoplast Surface Labeling with the 75 kD Anti-

serum. In order to obtain additional evidence that the 75 kDantiserum was directed against epitopes present on the plasmamembrane, an immunofluorescence assay was used. Becauseanimal IgG fractions were shown to bind to plant protoplastsurfaces-(21, 26, 28), it was first necessary to establish conditionswhere there was no background fluorescence. It was determinedthat nonspecific binding of either the primary or secondaryantibody was eliminated by first "blocking" the protoplasts withgoat IgG protein. By using a rhodamine-conjugated secondaryantibody, the problem of autofluorescence was completely cir-

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92.5-

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FIG. 6. Specificity of 75 kD antiserum against proteins from soybean root plasma membrane after two-dimensional gel electrophoresis. A, Amidoblack stain; B, stained with the 75 kD antiserum (200 ug membrane protein loaded).

pH 7.0

92.5

66.2 O °

pH 6.0 pH 5.0

I00 0 0 2O01313

FIG. 7. Comparison of proteins recognized by the 75 kD antiserumin tomato fruit plasma membrane and soybean root plasma membrane.Rectangles indicate proteins recognized in tomato, circles indicate pro-teins recognized on soybean root. The photographic images of Figures 2and 6 were xerographically enlarged to permit more detail in this figure.Only that portion of the gel staining positively is shown.

cumvented.Figure 4 demonstrates that the 75 kD antiserum was effective

as a protoplast surface label. When preimmune serum was usedas the primary antibody, no surface fluorescence was observed(Fig. 4). This result indicated that the 75 kD antiserum wasdirected against plasma membrane epitopes.75 kD Antiserum Recognizes Epitopes Present in Both Corn

Root and Soybean Root Membrane Fractions. To determinewhether the 75 kD antiserum recognized epitopes present inplasma membrane fractions from other plants, a 34/45% sucroseinterface was collected from corn root (a monocotyledonousplant) and soybean root (a dicotyledonous plant). Figure 5 showsthat the 75 kD antiserum from tomato does detect a 66 kDepitope in membrane fractions from both corn and soybean root.In soybean root membranes, a 75 kD band was also faintlystained. Hence, the 75 kD protein group appears to be a generalplant membrane epitope, common to both dicotyledonous andmonocotyledonous plants.To characterize further these common epitopes in soybean, a

two-dimensional gel was used. Figure 6B demonstrates that the75 kD group ofproteins is common to both tomato and soybean.An overlay figure was prepared from both tomato and soybeantwo-dimensional gels and is shown in Figure 7. In soybeans,there appeared to be a second, minor set of proteins that were

recognized by the 75 kD antiserum (Fig. 6B). This minor set ofproteins was seen as a single Mr of 65,600 but having a wide plrange-between pH 4.5 and 6.5. Two of these proteins were alsoseen in two-dimensional gels of proteins from tomato plasmamembranes, and are indicated in Figure 7. Because of the over-lapping Mr values between these two apparent sets of proteins, itis difficult to definitively state whether this expanded pl series insoybean constitutes a distinct class or whether it is a subset ofthe major 75 kD protein group.

DISCUSSION

A major protein group from the plasma membrane fraction oftomato cells was identified and studied by the use ofa polyclonalantiserum. This 75 kD protein group, and the antiserum to it,are of interest for several reasons. First, this protein group is oneofthe major proteinaceous constituents ofthe plasma membranefrom tomato cells. Second, it is also a component of membranefractions from both corn and soybean roots. Third, the 75 kDprotein group does not bind ConA and the antiserum appears tobind proteinaceous epitopes.The 75 kD protein group is a very abundant protein group in

the plasma membrane from tomato suspension cells. Plasmamembrane has been isolated from this source many times and itwas observed that the staining intensity of the 75 kD proteingroup varies between 10 and 50% ofthe total plasma membraneprotein (HD Grimes, unpublished data). The factors controllingthis alteration of the amount of the 75 kD protein group areunknown. This protein group is also a major component ofplasma membrane from tomato fruit (Fig. 2) and tomato roots(2) but the proportion of this protein group in the plasmamembrane of these tissues does not appear to change. These dataindicate that the 75 kD protein group, with its large mass andhigh concentration, is one of the major protein components ofthe tomato plasma membrane.The antigenic determinants of the 75 kD antiserum has been

referred to consistently in this paper as the '75 kD protein group,'because immunoblots of two-dimensional gels of proteins from

pH 6 pH 5 pH 7 pH 6

4

pH 5

0

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- 92.5

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- 31

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tomato plasma membranes primarily showed

group. However, the 75 kD antiserum may

protein sets, especially in plasma membranecorn

and soybean roots. In the two-dimensional

soybean plasma membrane-enriched fraction,

be two sets of reactive spots. The first set,

'major' set, correlated strongly with the

group-there was identical overlap in both Mr plrations. A second, 'minor' set may also

soybean set consists of about 8 to 10 polypeptides

but with a pH range between 4.5 and 6.5.

tides were shown to be identical to two

membrane. These spots may represent a

protein set recognized by the tomato

major set, however, shows a broad Mr range narrow

pH range. Because the Me's of these two

overlap, it is difficult to determine whether

protein sets or whether a single protein

two-dimensional gel analysis of protein from

membrane indicated predominantly one

chosen to refer to these epitopes as the

Another interesting feature of the 75 kD

it is common to plants of diverse taxonomic

feature suggests that the 75 kD protein

some essential function that plant membranes

Norman et aL. (24) have reported that the

that they generated to proteins of tobacco

cross-reactive with many species.

In order to identify and isolate the genes

membrane protein synthesis, it is imperative

recognize the peptide portion of the molecule

translationally added glycosyl chains. One

75 kD protein group was originally

production was that it did not appear

while many other plasma membrane proteins

with ConA. During this work, it was important

the epitopes being recognized by the 75

exclusively a glycosyl chain. To establish

serum recognized peptide epitopes, the

membrane fraction were either first

chemically deglycosylated with trifluoroacetic

probed with the 75 kD antiserum. Since

bound strongly after these treatments, it

kD antiserum probably recognizes peptide

protein group and is therefore a protein

for further studies of the molecular genetics

membrane.

LITERATURE CITED

1. ANDERSON RL, PM RAY 1981 Labeling

by a surface-localized glucan synthetase.

2. ANTHON GE, RM SPANSWICK 1986

H+-translocating ATPase from the plasma

Physiol 81: 1080-1085

3. BARTLES JR, LT BRAITERMAN, AL HUBBARD

domain markers of the rat hepatocyte J1126-1138

4. Booz ML, RL TRAVIS 1980 Electrophoretic

enriched plasma membrane fractions from

Physiol 66: 1037-10435. BRISKEN DP, RI POOLE 1983 Evidence,8-aspartyl

the phosphorylated intermediate of the

Plant Physiol 72: 1133-11356. DEBLAs AL, RO Kuuis, HM CHERWINSKI 1984 Mammalian brain antigen

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by affinoblotting with concanavalin A-peroxidase and treatment of the blotswith glycosidases. Anal Biochem 149: 218-224

9. GAMBORG CG 1981 Membrane glycoproteins and glycolipids: structure, local-ization and function of the carbohydrate. In JB Finean, RH Michell, eds,Membrane Structure. Elsevier/North Holland Biomedical Press, Amster-dam, pp 127-160

10. GOGSTAD GO, MB KRuTNEs 1982 Measurement of protein in cell suspensionsusing the Coomassie brilliant blue dye-binding assay. Anal Biochem 126:355-359

11. GRIMES HD, NM WATANABE, RW BREIDENBACH 1986 Plasma membraneisolated with a defined orientation used to investigate protein topography.Biochim Biophys Acta 862: 165-177

12. HoDGEs TK, RT LEONARD 1974 Purification of a plasma membrane boundATPase from plant roots. Methods Enzymol 32: 392-406

13. HODGEs TK, D MILLS 1986 Isolation of the plasma membrane. MethodsEnzymol 1 8: 41-54

14. HUBBARD AL, JR BARTLES, LT BRAITERMAN 1985 Identification of rat hepa-tocyte plasma membrane proteins using monoclonal antibodies. J Cell Biol100: 1115-1125

15. HUGHES RC 1976 Membrane Glycoproteins. Butterworths, London16. HUGHEs EN,JT AUGUST 1981 Characterization of plasma membrane proteins

identified by monoclonal antibodies. J Biol Chem 256: 664-67117. HUGHES EN,JT AUGUST 1982 Murine cell surface glycoproteins. Identification,

purification, and characterization of a major glycosylated component of1

10,000 daltons by use of a monoclonal antibody.J Biol Chem 257: 3970-3977

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