lsolation and ldentification of Ripening-Related Tomato Fruit ... - NCBI

Plant Physiol. (1 996) 1 1O : 875-882

lsolation and ldentification of Ripening-Related Tomato Fruit Carboxypeptidase Roshni A. M e h t a and Autar K. Mattoo* Plant Molecular Biology Laboratory, Beltsville Agricultura1 Research Center, United States Department of Agriculture/Agricultural Research Service, Beltsville, Maryland 20705-2350 (R.A.M., A.K.M.); and Department of Biological Sciences, University of Maryland, Catonsville, Maryland 21 228 (R.A.M.)

and exoproteases. Major exoproteases studied belong to Ser-type carboxypeptidases (Mikola and Mikola, 1986). Since protein turnover is a norm during fruit ripening/ senescence (Mehta et al., 1991; Mehta, 1993), proteases should play a role in these processes. Relatively low activities of proteolytic enzymes have been found in most fruits; however, these low activities may be sufficient for the intrinsic requirement of fruit ripening (Boller, 1986). A carboxypeptidase activity was detected in ripening tomato (Matoba and Doi, 1974).Subsequently, such an activity was shown to increase systemically in distant leaves upon wounding (Walker-Simmons and Ryan, 1977). Also, the gene encoding Leu aminopeptidase was cloned from tomato and was suggested to function as a component in defense response (Pautot et al., 1993).More work is needed to identify and characterize specific proteases that are upor down-regulated during ripening to study their role(s) in the ripening/senescence process. Carboxypeptidases are relatively abundant in most tissues of a11 higher plants, monocots, dicots, and gymnosperms (Zuber, 1964; Wells, 1965; Ihle and Dure, 1972; Matoba and Doi, 1974; Doi et al., 1980; Mikola, 1983; Mikola and Mikola, 1984; Winspear et al., 1984). They have been proposed to play an important role, relative to endopeptidases, in the hydrolysis of reserve proteins (Mikola, 1983). We have confirmed the presence of carboxypeptidase in different organs of the tomato plant, have shown its development-specific induction upon wounding, and have established its location in the cellular vacuoles of the pericarp tissue using immunogold EM (Mehta et al., 1996).Here we report purification and identification of the ripening-related tomato fruit vacuolar carboxypeptidase.

Tomato (Lycopersicon esculentum) fruit carboxypeptidase active on N-carbobenzoxy Z-i-phenylalanine-i-alanine was found to constitute a family of isoforms whose abundance changed differentially during ripening. A specific polyclonal antibody against the fruit carboxypeptidase was raised in rabbits and used to purify and identify the protein. The data from immunoaffinity chromatography, immunoinhibition studies, immunoprecipitation of the in vivoand in vitro-labeled proteins, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of native isoforms strongly suggest that the fruit carboxypeptidases are monomers or oligomers of 68- and/or 43-kD subunits.

Protein turnover and nitrogen mobilization are important components of dynamic cellular metabolism during growth, development, and ripening of fruits (Brady, 1987; Brady and Spiers, 1991). For instance, dedifferentiation of chloroplasts to chromoplasts during tomato (Lycopersicon esculentum) ripening involves a decline in the amounts of many soluble and membrane-associated plastid proteins while new chromoplast-specific proteins accumulate (Bathgate et al., 1985; Oren-Shamir et al., 1993). Differential protein synthesis in a ripening fruit was demonstrated from earlier studies using incorporation of precursors into proteins in tomato fruit tissue slices (Baker et al., 1985) and estimating recovery of polysomes from intact avocado fruit (Tucker and Laties, 1984).The observed changes in mRNA and protein populations (Mehta et al., 1991) and in specific transcripts (Gray et al., 1992) indicate that macromolecular machinery for the production of enzymes essential for ripening remains intact and active and that ripening involves substantial turnover of existing and newly synthesized proteins. Although considerable literature has accumulated concerning gene expression during ripening of fruits, little is known about turnover of proteins during this process. Most of the published work concerning plant protein turnover has dealt with the breakdown of storage proteins during maturation and germination of seeds (Preston and Kruger, 1986; Wilson, 1986) and the light-mediated degradation of chloroplast membrane proteins (Mattoo et al., 198913). Proteolysis in such cases seems to involve endo-

MATERIALS AND METHODS Plant Material

Tomato (Lycopersicon esculentum cv Pik-red) plants were grown either in a greenhouse under 16 h of daylight and 8 h of darkness at temperatures ranging from 25 to 30°C or in the U.S. Department of Agriculture fields at Beltsville, MD. Unblemished, uniformly shaped fruit at the pink (early red) stage were harvested, surface sterilized with 70% ethanol, and then thoroughly rinsed with sterile water. The fruits were

* Corresponding author; e-mail900422@~mdd.umd.edu; fax 1-301504-5320.

Abbreviations: FPLC, fast protein liquid chromatography; TNBS, trinitrobenzene sulfonic acid. 875

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sliced, and the pericarp tissue was cut into smaller (1-5 cm) pieces and stored at -80°C until further use.

I

1

Purification of Tomato Fruit Carboxypeptidase Preparation of Crude Extracts

The following procedures were conducted at O to 4°C. Total proteins were extracted using the method of WalkerSimmons and Ryan (1980) with minor modifications. Tomato fruit tissue was homogenized with 2 volumes of extraction buffer (10 mM acetate buffer, pH 5.2, 1 mM EDTA, 0.1 M SUC,1 pg/mL leupeptin, 1 pg/mL pepstatin, and 0.1% p-mercaptoethanol) in a Waring commercial blender.' The homogenate was squeezed through severa1 layers of cheesecloth and filtered through a single layer of Miracloth (Calbiochem).

O

20

10

30

Elution Volume (ml)

Ammonium Sulfate Fractionation and Dialysis

The crude extract was centrifuged at 5,OOOg for 10 min, and the proteins in the supernatant that precipitated between 40 and 80% ammonium sulfate saturation (containing essentially a11 of the carboxypeptidase activity) were collected by centrifugation at 10,OOOg for 30 min. The resuspended 40 to 80% ammonium sulfate precipitate was dialyzed against 2 X 4-L changes of the extraction buffer lacking p-mercaptoethanol. lon-Exchange Chromatography

Further enrichment of carboxypeptidase activity was achieved on an FPLC Mono-S HR 5/5 ion-exchange column (Pharmacia)equilibrated with 10 mM sodium acetate, pH 5.2, 1 mM EDTA. The dialyzed ammonium sulfate-precipitated proteins were loaded at a flow rate of 0.5 mL/min. Elution of carboxypeptidasewas achieved with 25 mL of a linear gradient of O to 0.5 M NaCl. Fractions (0.5 mL) were collected and checked for enzyme activity (Fig. 1).A,,, was used to estimate protein concentration (Fig. 1).The fractions containing peak enzyme activity were pooled and concentrated by precipitation with ammonium sulfate (to 80% saturation), followed by centrifugation at 10,OOOg for 30 min. The precipitates were resuspended in 10 mM acetate buffer, pH 5.2, and dialyzed ovemight against the same buffer. Determination of Molecular Weight

The apparent molecular weight of carboxypeptidase was estimated by chromatography on a Superose 12 HR column (10 X 30 cm, FPLC, Pharmacia) equilibrated with 0.2 M sodium acetate, pH 5.2, l mM EDTA, 0.2 M SUC.Two hundred microliters of Mono-S-purified carboxypeptidase fraction were loaded onto the column. The proteins were eluted at a flow rate of 0.5 mL/min and the fractions were assayed for protein content and enzyme activity (Fig. 2). In separate runs,

' Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the products by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Figure 1. Mono-S chromatography of tomato fruit carboxypeptidase. The ammonium sulfate-fractionated pink fruit extract (5 mg of protein) was chromatographed on a Mono-S column (FPLC, Pharmacia) equilibrated with 10 mM sodium acetate, pH 5.2, 1 mM EDTA. Carboxypeptidase activity was assayed in 0.05-mL aliquots of each fraction (A4,,J. A,,, (protein) and the stepwise gradient of NaCl

(broken line) are also indicated.

blue dextran (V, marker), catalase, aldolase, BSA, and chymotrypsinogen (Pharmacia) were loaded individually onto the column to calculate'K, values. The location of the standards in the eluted fractions was determined by A,,,. Carboxypeptidase Assay

Carboxypeptidase activity was routinely assayed with the substrate N-carbobenzoxy-Z-L-Phe-L-Ala at 30°C as described previously (Salgo and Feller, 1987). N-Carbobenzoxy-Z-L-Phe-L-Ala (200 pmol) was dissolved in 2 mL of DMSO and made to a final volume of 100 mL with 50 mM acetate buffer, pH 5.0, containing 0.5 mM EDTA (stock substrate solution). The reaction mixture (0.4-mL final volume) containing 0.1 mL of substrate, various aliquots (0.7-50 pg) of enzyme protein, and the acetate buffer was incubated at 30°C for 1 h. The controls were prepared without the substrate. The reaction was stopped by the addition of 1 mL of freshly prepared TNBS solution (10 mg of TNBS in 30 mL of 50 mM borate buffer, pH 9.5'1, and the samples were further incubated for 1 h in the dark. The liberated C-terminal amino acid reacted with TNBS to yield a yellow product, which was measured at 405 nm with a multichannel spectrophotometer. The absorbance differences were converted to micromoles Ala produced using a calibration curve with L-Ala (up to 50 nmol). One unit of carboxypeptidase activity is expressed as one /*.mole of L-Ala released per hour. Specific activity is given as units per milligram of protein. Production of Antibodies

Antiserum to purified leaf carboxypeptidase raised in mice was a gift from Dr. M.K. Walker-Simmons (Washington State University, Pullman). The serum was precipitated

Purification of Tomato Fruit Carboxypeptidase

Elution Volume (ml) Figure 2. Determination of molecular weight of the native tomato fruit carboxypeptidase. The Mono-S-fractionatedenzyme was loaded on to a gel filtration column (FPLC, Superose 12). Proteins were eluted in 200 mM sodium acetate, pH 5.2, 1 m M EDTA, 200 mM Suc at a flow rate of 0.5 mL/min at 4°C. Fractions were assayed for carboxypeptidase activity (A4o5).Molecular weights of 130,000 and 67,000 were estimated by comparison to standard protein markers indicated by arrows in the following sequence: catalase (molecular weight 232,000),aldolase (molecularweight 158,000), BSA (molecular weight 67,000), and chymotrypsinogen (molecular weight 25,000) (Pharmacia).

with ammonium sulfate (25-50% saturation), resuspended in PBS (10 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl), and dialyzed overnight against the same solution. The antibodies were then applied to a Protein A-Sepharose CL-4B column (FPLC, Pharmacia). Adsorbed immunoglobulins were eluted with 0.1 M citrate, pH 6.0, neutralized with 5 N NaOH, and then dialyzed against PBS. The antibodies were stored in capped vials at -80°C. This antibody to leaf carboxypeptidase cross-reacted strongly with a 43-kD protein band on immunoblots of tomato fruit extract resolved by SDS-PAGE. The 43-kD protein band was cut out from SDS-polyacrylamide gels and used together with 50 pg of protein from the carboxypeptidaseactive Mono-S fraction to generate fruit carboxypeptidasespecific antisera in rabbit. Immunization consisted of a total of four injections, approximately every 2 weeks, over a 2-month period. The antiserum titer was checked periodically by spotting the tomato fruit and leaf extracts on nitrocellulose paper and incubating with serially diluted antisera. The protocol was the same as that for developing an immunoblot. The rabbits were killed after 2.5 months when the titer was deemed appropriate. The antibody and preimmune serum (collected before the first antigen injection) were purified and stored as described above. lmmunoprecipitation of Carboxypeptidase Activity and Protein

Immunoinhibition of tomato carboxypeptidase activity was determined by incubating a series of 5 to 25 enzyme

a77

units in 250-pL aliquots with increasing amounts of the anti-carboxypeptidase antibodies diluted in PBS (pH 8.0) for 4 to 16 h at 4°C. The precipitates obtained were centrifuged at l0,OOOg for 10 min at 4°C. The supernatants were collected and assayed for residual enzyme activity. Control tubes contained preimmune serum instead of the anticarboxypeptidase antibodies. Protein A-Sepharose CL-4B (50 pL, Pharmacia) was then added to each tube, the tubes were centrifuged at l0,OOOg for 5 min, and the supernatant was assayed for residual enzyme activity. Ammonium sulfate precipitates from pink fruit extract (100 pL) were used to immunoprecipitate carboxypeptidase protein by the procedure described by Harlow and Lane (1988) with some modifications. The precipitates were first adjusted to a 2% final concentration of SDS and heated to 100°C for 1 min. After the samples were cooled to 4"C, 50 pL of 4X immunoprecipitation buffer ( 1 X = 50 mM TrisHC1, pH 7.4, 1%Triton X-100,0.5 M NaC1,2% BSA) and 100 pL of Protein A-Sepharose (10%, v/v, in immunoprecipitation buffer) were then added and the mixture was incubated for 1 h at 4°C' with gentle rocking. The Sepharose beads were pelleted by centrifugation at l0,OOOg for 15 s. To the supernatant, 1 pL of preimmune serum was added and the mixture was incubated for 1 h at 4°C. Protein A-Sepharose (100 pL) was then added and the mixture was further incubated for 1h. The pellets were collected by centrifugation at l0,OOOg for 5 min and the supernatants were transferred to new tubes. Subsequently, anti-carboxypeptidase antibody (2 pL) was added and immunoprecipitates were collected as described above. The immunoprecipitates were washed five times with 1X immunoprecipitation buffer, twice with 1 M NaCl, and once with double-distilled water. Finally, 50 pL of sample application buffer (Mattoo et al., 1981) were added to the washed pellet, the mixture was heated to 90°C for 3 min followed by brief centrifugation to remove the beads, and the supernatant was then analyzed by SDS-PAGE (Laemmli, 1970). lmmunoaffinity Purification

Immunoaffinity matrix was prepared by coupling purified polyclonal anti-carboxypeptidase antibodies to cyanogen bromide-activated Sepharose 4B (Pharmacia) following the instructions of the supplier. Ammonium sulfate-precipitated pink fruit extract was centrifuged at 10,OOOg for 30 min to remove particulate matter and then loaded onto the column previously equilibrated with binding buffer (10 mM phosphate, pH 7.4). The mixture was rotated on a nutator for 1h at 4°C and then passed through the affinitymatrix column at a flow rate of approximately 2 mL/h. The column was washed with 20 bed volumes of binding buffer. Carboxypeptidase activity was eluted from the column with 100 mM Gly, pH 2.2. Fractions (1 mL) were collected, the pH of each adjusted to 5.2 with dilute NaOH, and then dialyzed against 10 mM sodium acetate, pH 5.2,l mM EDTA to remove Gly prior to assaying for carboxypeptidase activity. Enzymatically active fractions were pooled, freeze dried, resuspended in 10 mM sodium acetate, pH 5.2, 1 mM EDTA buffer, and prepared for SDS-PAGE and immunoblotting.

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Mehta and Mattoo

878 Cel Electrophoresis and lmmunoblotting

Protein content of the ammonium sulfate-precipitated tomato fruit extracts was measured using Rose Bengal dye as described by Elliott and Brewer (1978). For denaturing SDS-PAGE, soluble proteins were denatured in sample application buffer (Mattoo et al., 1981) and heated at 90°C for 3 min before fractionation on 10% (w/v) polyacrylamide gels (Laemmli, 1970). The gels were stained with 0.05% (w/v) Coomassie brilliant blue R-250 or silver (Wray et al., 1981). Molecular masses were determined by comparison to standard molecular mass markers ranging from 14 to 200 kD (Amersham). For native PAGE, samples of tomato fruit extracts were prepared with 14% SUC,0.1% bromphenol blue and applied to nondenaturing 10% polyacrylamide gels. The gels and electrophoresis buffer were exactly as for SDS-PAGE except that SDS was omitted and electrophoresis was conducted at 4°C. Immunoblotting was performed essentially as described by Towbin et al. (1979). Proteins were transferred electrophoretically overnight at 30 V (approximately 200 mA) to nitrocellulose paper (BA 85; Schleicher & Schuell) in 25 mM Tris, 192 mM Gly, pH 8.3, 20% (v/v) methanol, and 0.02% (w/v) SDS for denaturing gels. Nondenaturing gels were transferred onto nitrocellulose paper using the transfer buffer lacking SDS and a current of 30 mA for 16 h at 5 to 10°C. Immunoblot analysis was performed as described by Callahan et al. (1989).

Plant Physiol. Vol. 110, 1996

fuged for 5 min prior to use. Immunoprecipitation was performed according to the method of Mattoo et al. (1989a). The pellets obtained were analyzed by SDS-PAGE and fluorography as described by Mehta et al. (1991). RESULTS Characteristics of Tomato Fruit Carboxypeptidase

During our initial attempts to purify the fruit carboxypeptidase, we observed that the activity was highly labile in cell extracts, but once precipitated with ammonium sulfate (40-80%) the enzyme could be stored at 0°C for 2 to 3 d or at -80°C for up to 4 years without significant loss of activity. Improved stability and recovery during purification were obtained upon inclusion of protease inhibitors such as leupeptin (1pg/mL), pepstatin (I pg/mL), and P-mercaptoethanol (0.1%) in the extraction buffer. Purification of the carboxypeptidase activity froin tomato fruit using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration as described in ”Materials and Methods” resulted in 200-fold enrichment of the enzyme activity, but large losses occurred following gel filtration (Table I). Prominent among the several polypeptides enriched was a 43-kD band as revealed by SDS-PAGE (data not shown). Gel filtration experiments showed that the enriched carboxypeptidase activity was composed of two proteins with molecular weights of 130,000 and 67,000 (Fig. 2). However, the purification method used gave poor yields and resulted in the inactivation of the enzyme even though buffers contained several different inhibitors of In Vivo-Labeling Experiments proteolytic activity (Table I). Therefore, an alternative approach using affinity chromatography with carboxypeptiTomato fruit pericarp tissue was cut into small pieces and placed in a vacuum chamber and 100 pCi of ~ - [ ~ ~ S l M e t dase-specific antibody bound to a matrix was attempted. (New England Nuclear, specific activity 1134 Ci/mmol) per 5 g of tissue were vacuum infiltrated (30 cm Hg) for 1 Specificity of Antibodies to Fruit min. The vacuum was released and labeling continued for Carboxypeptidase and lmmunoprecipitation 3 h. Soluble proteins were extracted as described before (Mehta et al., 1991) and subjected to immunoprecipitation Polyclonal antibodies were raised against tl-ie tomato with anti-carboxypeptidase antisera as described below. fruit carboxypeptidase, generated as described in ”Materials and Methods.” These antibodies immunoinhibited carboxypeptidase activity in fruit extracts. Figure 3 depicts the In Vitro Translation and lmmunoprecipitation ability of the antibody to react with the enzyme protein with resultant loss of enzyme activity from the supernaIn vitro translations were performed in the presence of tant. The antibody precipitated 51% of the fruit car50 pCi of ~ - [ ~ ~ S l M (Amersham) et and 1.5 pg of total RNA boxypeptidase activity. Preimmune rabbit serum did not isolated from breaker tomato fruit (Mehta et al., 1991) using inhibit or immunoprecipitate the enzyme activity. rabbit reticulocyte lysate (Amersham) in 50 p L (final volTo identify the proteins associated with the immunopreume) according to the supplier’s instructions. Translation cipitation of carboxypeptidase activity, the immunopreproducts used for immunoprecipitation were centrifuged cipitates (from pink tomato fruit extract treated with for 5 min in an Eppendorf microfuge to remove insoluble anti-fruit-carboxypeptidase antibody) were washed, and material. Preimmune and immune sera were also centriTable 1. Purification of carboxypeptidase from tomato fruit Purification Ster,

Enzyme Activity pmol h-

Crude extract Ammonium sulfate (40-80%) Mono-S (ion exchange) Superose 12 (gel filtration)

1094

508 337 6.9

’

Total Protein mg

2670 141 6.57 0.08

Specific Activity pmol

h-

’ mg0.41 3.6 51.4 86

protein

Purification

Recovery

fold 1 8 125 209

76 1O0 46 31 0.63

Purification of Tomato Fruit Carboxypeptidase

879

125

•a

100

kD ,

M

75 -

97.4 -

s.

68 50 -

-OF

43-

3

T>

25 -

2910

20

30

Antibody (|ig)

18 -

Figure 3. Immunotitration of Carboxypeptidase activity from tomato fruit extracts. Tomato fruit extract (0.25 ml) was incubated with increasing amounts of either anti-carboxypeptidase antibody (F) or preimmune serum (C) for 4 to 16 h at 30°C in 0.2 M phosphate buffer, pH 6.5. Fifty microliters of Protein A-Sepharose CL-4B (57 ng/mL) were then added to each tube. The tubes were further incubated for 1 h and centrifuged, and the supernatant fraction was assayed for residual Carboxypeptidase activity.

Figure 4. Immunoprecipitation of Carboxypeptidase from pink tomato fruit. Immunoprecipitations were carried out using Protein A-Sepharose CL-4B as described in the text. Immunopellets obtained with normal rabbit serum (lane 3) and the immune serum (lane 4) were fractionated alongside the untreated 40 to 80% ammonium sulfate precipitate of pink tomato fruit extract (lane 2). Positions of standard protein markers (lane 1) are indicated in kD.

associated proteins were solubilized and fractionated by SDS-PAGE. The polyacrylamide gels were stained with Coomassie blue to reveal the proteins (Fig. 4). The immunoprecipitation protocol was used under high-stringency conditions to eliminate nonspecifically bound proteins. Evidently, two polypeptides of 68 and 43 kD were immunoprecipitated by the antibody (Fig. 4, lane 4) but not by the preimmune serum (Fig. 4, lane 3). Comparing the intensity of the stained bands in the control, nonimmunoprecipitated sample with those immunoprecipitated by the antibody shows that the two proteins are present in low abundance.

1.0- 00099-

kD

97.4 68 0.5-

8 I 2910

20

30

Elution Volume (ml)

40

M

Purification of Fruit Carboxypeptidase by Immunoaffinity Chromatography Anti-carboxypeptidase antibodies were coupled to cyanogen bromide-activated Sepharose CL-4B. Ammonium sulfate-fractionated pink fruit extract was applied to the affinity matrix. Carboxypeptidase was then eluted from the column with 0.1 M Gly, pH 2.2, without substantial loss of activity (Fig. 5 A). Upon SDS-PAGE under reducing conditions and subsequent staining of the gel, the active fractions were found to contain two proteins of 68 and 43 kD

1

2

Figure 5. A, Immunoaffinity chromatography of Carboxypeptidase from pink tomato fruit. Solubilized ammonium sulfate precipitate (0.5 ml) was applied to an anti-carboxypeptidase polyclonal antibody column (bed volume = 3.5 ml). After the column was washed with binding buffer (10 mM phosphate, pH 7.4), the bound enzyme was eluted with 0.1 M Gly-HCI, pH 2.2 (arrow). The flow rate was 2 ml_/h, and fractions of 1 ml were collected. Protein concentration was measured by A2Bn and Carboxypeptidase activity was measured in 25 (j-L of dialyzed fractions. B, An aliquot of the untreated 40 to 80% ammonium sulfate precipitate of pink tomato fruit extract (lane 1), immunopurified Carboxypeptidase (lane 2), and 0.5 /xg of standard protein markers (lane M) were electrophoresed on 10% SDS-polyacrylamide gel, which was then stained with silver nitrate.

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Mehta and Mattoo

(Fig. 5B). Thus, immunoinhibition of carboxypeptidase activity in cell-free extracts of pink tomato fruit correlates with not only immunoprecipitable 68- and 43-kD proteins but also with their co-purification using immunoaffinity chromatography. Immunoprecipitation of in Vivo-Labeled and in VitroTranslated Carboxypeptidase

Further confirmation and identification of carboxypeptidase protein were approached by analyzing immunoprecipitates from radiolabeled fruit tissue and in vitro-translated breaker fruit poly(A) + RNA. Results presented in Figure 6 indicate that the antibody selectively immunoprecipitates a radioactive protein of 68 kD (Fig. 6B) from the radiolabeled fruit extract that co-electrophoresed with a 68-kD protein from the in vitro-translation mixture (Fig. 6A, lane 4). Four other proteins of 91, 51, 39, and 31 kD were additionally present in the immunoprecipitates of the in vitro-translation products. The 68-kD protein is common

A

In Vitro

B In Vivo

kD 92.5-

69 — 46 —

30-

14.31

3

4

5

6

7

Figure 6. Comparison of immunoprecipitated proteins from in vitrotranslation products and from in vivo-labeled tomato fruit proteins. Poly(A) + RNA was isolated from green tomato fruit and translated in vitro using rabbit reticulocyte lysate as described in the text (A, lane 2). Proteins from green tomato fruit were labeled in vivo with [ 35 S]Met (B, lane 5). All of the samples were treated with anticarboxypeptidase antibody or preimmune serum and the immunoprecipitates that were recovered were fractionated on polyacrylamide gels. The radioactive protein bands were detected by fluorography. Lane 1, Reticulocyte lysate without added RNA; lane 2, translation products from RNA isolated from green fruit; lanes 3 and 6, immunoprecipitates obtained with preimmune serum; lanes 4 and 7, immunoprecipitates obtained with anti-carboxypeptidase antibody; lane 5, untreated [35S]Met-labeled sample. Radioactive marker proteins are shown on the left.

Plant Physiol. Vol. 110, 1996

to immunoprecipitates from in vivo- (both unlabeled and radiolabeled) and in vitro-labeled tomato proteins, suggesting its identity as a fruit carboxypeptidase. It is possible that the 91-kD band could be a precursor that is processed to the 68-kD form. Likewise, the 51-kD in vitroproduct could be a precursor for the 43-kD carboxypeptidase, which, for some unknown reason, was not detected in these experiments. It is possible that the 43-kD polypeptide results from processing of a higher molecular weight protein. Processing of carboxypeptidase I from a single precursor polypeptide chain into two subunits has been reported for the enzyme from germinated barley and carboxypeptidase Y (Mikola and Mikola, 1986). Alternatively, the 91-, 51-, 39-, and 31-kD protein bands may represent other novel carboxypeptidase proteins. The possibility that the low-molecular-mass 39- and 31-kD polypeptides may be the degradation products or early quitters during translation cannot be discounted. Fruit Carboxypeptidase Is Present in Isoforms

Preparative IEF of tomato fruit extracts revealed the presence of three carboxypeptidase isoforms fractionating at pis 4.36, 5.35, and 5.93 (Mehta, 1993). The apparent molecular weights of carboxypeptidases obtained upon gel filtration on Superose 12 column were 130,000 and 67,000 (Fig. 2). The presence of isoforms of carboxypeptidase in tomato fruit is consistent with other reports of carboxypeptidase isozymes in barley (Mikola, 1983; Dal Degan et al., 1994), wheat (Mikola and Mikola, 1984), rice (Doi et al., 1980), and watermelon (Matoba and Doi, 1975). For further confirmation and finer analysis, the isoforms of carboxypeptidase were analyzed on native gels. Tomato fruit extracts fractionated on native gels were immunoblotted and the blots probed with the anti-carboxypeptidase antibody. Four immunoreactive bands were consistently observed on the blots and these were designated Fl, F2, F3, and F4 (Fig. 7A). To ascertain the subunit composition of these isoforms, each band from native gels was excised, subjected to SDS-PAGE, and immunoblotted (Fig. 7B). The Fl and F2 isoforms appeared to be made up solely of the 68-kD protein band. The F3 isoform showed the presence of both 68- and 43-kD proteins. The F4 band was made up solely of the 43-kD band. These results indicate that the 68and 43-kD polypeptides are the two main carboxypeptidase polypeptides present in tomato fruit. Carboxypeptidase Isoform Pattern during Ripening

The accumulation of the various carboxypeptidase isoforms in tomato fruit was analyzed at different stages of ripening and the results are presented in Figure 8. The most obvious change seen was the absence of F2 isoform and the presence of F3 isoform solely in the red fruit. The Fl protein was faintly visible in the green and breaker fruits, increasing in intensity in pink and red fruit. The level of the F2 isoform was more or less similiar at all stages, except in red, where it was absent. The F4 isoform was present at all stages, with the maximum level in the pink fruit (Fig. 8). These data reveal a differential pattern of accumulation of

881

Purification of Tomato Fruit Carboxypeptidase

the four Carboxypeptidase isoforms in the fruit during ripening.

The present study identified ripening-related carboxypeptidases from tomato fruit. The generation of a polyclonal antibody against this enzyme that reacted with native Carboxypeptidase enabled us to successfully use it in the preparation of an affinity column and the detection of 68- and 43-kD forms of Carboxypeptidase. The identity of the 68- and 43-kD proteins as fruit carboxypeptidases is suggested by their co-purification on immunoaffinity chromatography, their exclusive presence in the immunoprecipitates of fruit extract proteins, and their presence as constituents of native Carboxypeptidase isoforms. The identity of the 68-kD Carboxypeptidase was further confirmed by immunotitration and immunoprecipitation of in vivo-synthesized and in vitro-translated proteins. The absence of a 43-kD protein band in the immunoprecipitates of in vivo-labeled tomato fruit extracts and in vitro-translated protein products raises the possibility that this protein either is synthesized at lower rates or is a processed form of a seemingly higher molecular weight protein. The presence of the 91- and 51-kD protein bands in immunoprecipitates of in vitro-translated proteins suggests that they may be the precursors to the 68- and 43-kD subunits, respectively. Synthesis of a precursor form of Carboxypeptidase and its processing to mature, low-molecular-weight form has been reported for the yeast carboxypeptidase Y (Vails et al., 1990) and mammalian lysosomal carboxypeptidases A, B, and C (Mikola and Mikola, 1986). Carboxypeptidase I from germinated barley has been shown to consist of two polypeptide chains that arise

B Denaturing PAGE

PAGE

kD

132-

66-

kD

*

-F2 -F3 -F4

I

Fl

M

kD

DISCUSSION

Native

STAIN

F2

F3

F4

68 43

Figure 7. Isozymes of tomato fruit Carboxypeptidase and identification of their subunits. Cell-free extract from pink fruit was fractionated on native, nonreducing polyacrylamide gels and electrotransferred onto nitrocellulose paper. The immunoblot was then developed with anti-carboxypeptidase antibodies (A). The consistently observed cross-reactive protein bands (F1-F4) were then excised, re-electrophoresed under denaturing conditions, and immunoblotted. Each blot was then developed with anti-carboxypeptidase antibodies (B). Asterisks on the right indicate the cross-reactive subunit polypeptide.

G

B

P - F1

132-

66-

Figure 8. Immunoblot analysis of Carboxypeptidase isozymes in tomato fruit at different stages of ripening. Tomato fruit extracts from green (G), breaker (B), pink (P), and red (R) fruit were fractionated on native polyacrylamide gels. The gels were either stained with silver nitrate (STAIN) or blotted onto nitrocellulose paper and reacted with anti-carboxypeptidase antibody (BLOT). Molecular weight markers are shown on the left and the positions of consistently observed immunoreactive protein bands Fl to F4 are also indicated.

by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain (Doan and Fincher, 1988). We also considered the possibility that the fruit carboxypeptidases may be glycosylated, as has been reported for Carboxypeptidase Y, wherein the carbohydrate moiety contributes 10 kD to the 57-kD polypeptide backbone of Carboxypeptidase Y (Vails et al., 1990). However, using Con-A Sepharose chromatography, the 43-kD form, and not the 68-kD form, was found to be glycosylated (Mehta, 1993). The presence of Carboxypeptidase isoforms in tomato fruit was ascertained from gel filtration (Fig. 2), immunoblot analysis of cell-free extracts fractionated on native gels (Fig. 7), and IFF experiments (Mehta, 1993). Analysis of the Carboxypeptidase isoforms from pink tomato fruit by SDSPAGE revealed that the various isozymes were made up of either the 68-kD protein, the 43-kD protein, or both. These data suggest that, like in barley (Mikola, 1983), the tomato fruit contains both monomeric and possibly polymeric forms of Carboxypeptidase. These results also suggest that the Carboxypeptidase isoforms are immunologically related and may share common structural properties. However, the differences in the time of appearance and relative levels of at least the four isozymes raise the possibility that more than one gene product may be responsible for carboxypeptidase expression during tomato fruit ripening. The identification and demonstration of different isoforms of Carboxypeptidase in tomato fruit raise additional questions concerning the developmental and environmental regulation of the enzyme. Are the different isoforms products of different genes? Are these isoforms interconvertible? Are the different isoforms regulated differentially by different stimuli? For instance, is wound-inducible Carboxypeptidase different from the developmentally regulated enzyme? To address these questions, it is important to isolate and characterize each isozyme independently of one another. It remains to be proven by comparison of the base sequences of the respective cDNAs whether there is more than one gene family coding for wound-induced and developmentally induced carboxypeptidases.

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ACKNOWLEDCMENTS

We thank Dr. M. Walker-Simmons for the gift of antiserum against tomato leaf carboxypeptidase; Dr. A.M. Mehta for helpful suggestions throughout the course of this work, and Drs. J.D. Anderson and M. Tucker for constructive comments on the original manuscript. We also thank members of the Mattoo group for a critica1 reading of the manuscript. Received May 26, 1995; accepted November 18, 1995. Copyright Clearance Center: 0032-0889/96/110/0875/08. LITERATURE ClTED

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