Purification of Tomato Sucrose Synthase Phosphorylated Isoforms by Fe(III)-Immobilized Metal Affinity Chromatography

Archives of Biochemistry and Biophysics Vol. 365, No. 1, May 1, pp. 163–169, 1999 Article ID abbi.1999.1146, available online at http://www.idealibrary.com on

Purification of Tomato Sucrose Synthase Phosphorylated Isoforms by Fe(III)-Immobilized Metal Affinity Chromatography Raphae¨l Anguenot, Serge Yelle, and Binh Nguyen-Quoc 1 Centre de Recherche en Horticulture, De´partement de phytologie, FSAA, Universite´ Laval, Sainte-Foy, Que´bec, Canada G1K 7P4

Received December 3, 1998, and in revised form February 3, 1999

The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report the in vitro phosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca 21dependent protein kinase activity. Two distinct 32Plabeled peptides detected in the tryptic peptide map of in vitro 32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major 32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two 32Plabeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major 32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform. © 1999 Academic Press Key Words: tomato fruit; sucrose synthase; protein phosphorylation; calcium; IMAC.

1 To whom correspondence should be addressed at Centre de Recherche en Horticulture, Pavillon de l’Envirotron, Universite´ Laval, Sainte-Foy (Que´bec), Canada G1K 7P4. Fax: (418) 656-7871. E-mail: [email protected].

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) 2 catalyzes the reversible conversion of sucrose and UDP to UDP-Glc and fructose. This reaction is considered to function in vivo in the sucrose-breakdown direction (1), providing substrates for actively growing tissues (2, 3) and sink storage organs (3– 6). In most higher plants, at least two differentially expressed gene families encode homologous SuSy isozymes constituting a tetrameric enzyme (7–9). Although in vitro modulation of SuSy activity by hexoses (3, 10), redox agents (11), and protein factors (12) has been reported, extractable SuSy activity is generally correlated with both transcript and protein levels (6, 10). However, recent reports showing that SuSy is phosphorylated in vivo (13, 14) have motivated investigations to understand the biochemical basis and the physiological importance of this posttranslational modification (15–18). Feeding maize shoots with [ 32P]Pi led to the phosphorylation of a seryl residue (-Leu-Ser-Arg-Leu-HisSer 15-Val-Arg-; Ser 15 being the phosphorylated residue) localized on the N-terminus extremity of the maize SuSy SS2 isozyme (15). This phosphorylation site, well conserved among plant species and SuSy isozymes, is the substrate of a 65-kDa maize Ca 21dependent protein kinase (CDPK) (15). Zhang and Chollet (16) have also partially purified a 55-kDa soybean nodule CDPK that phosphorylates both SuSy and phosphoenol-pyruvate in vitro. Possible physiological roles of this posttranslational modification of SuSy 2

Abbreviations used: SuSy, sucrose synthase; IMAC, immobilized metal affinity chromatography; CDPK, Ca 21-dependent protein kinase; TLE, thin-layer electrophoresis; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; FPLC, fast-protein liquid chromatography; IDA, iminodiacetic acid; CIP, calf intestinal phosphatase; PKA, protein kinase A; TFA, trifluoroacetic acid; EGTA, ethylene glycol bis(b-aminoethyl ether) N,N9-tetraacetic acid; Mes, 4-morpholineethanesulfonic acid. 163

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have emerged from studies on maize plants (15, 17). First, incubation of leaf maize extracts containing partially dephosphorylated SuSy with ATP lowered K m of SuSy for UDP (5-fold) and sucrose (2.5-fold) (15). However, activation of sucrose cleavage by ATP has been attributed in part to an artifactual UDP-glucose formation in crude extracts (17). Thus, modulation of the kinetic properties of SuSy by phosphorylation still remains to be determined with purified SuSy. More recently, Winter et al. (17) have observed that the partition of maize SuSy between the soluble and microsomal fractions of maize pulvinus extracts was modulated by a phosphorylation/dephosphorylation mechanism possibly involved in the graviresponse. Whereas the presence of a multigene family of SuSy has been described in both monocotyledonous and dicotyledonous plants (9, 19, 20), only one complete tomato SuSy mRNA (TOMSSF) is presently known (21). In situ hybridization of tomato fruit cryosections with a probe consisting in a fragment of a potato SuSy cDNA (POTSYN) 97% homologous to TOMSSF allowed the detection of SuSy mRNA in cells surrounding vascular bundles and starch-accumulating cells (6). Moreover, SuSy activity during the maximum fruit growth is well correlated with starch content (5, 22) and with tomato fruit weight at maturity (5). For these reasons, SuSy activity was proposed as a biochemical marker of sink strength in this organ. The recent findings suggesting the physiological importance of SuSy phosphorylation (15, 17) led us to question whether a similar posttranslational modification of SuSy could affect carbon metabolism and/or sink activity in tomato fruit. Surprisingly, the expected serine 11 residue related to the major phosphorylation site of maize SuSy was replaced by an arginine residue in the deduced sequence of TOMSSF (-Val-Leu-Thr-Val-His-Arg 11 -Leu-Arg). Given this fact, we tried to clarify whether tomato SuSy could undergo a posttranslational modification by phosphorylation. In this study, we report the in vitro Ca 21-dependent phosphorylation of tomato fruit SuSy by an endogenous protein kinase activity. To assess the effect of this chemical modification on the kinetic parameters of SuSy, metal affinity chromatography has been used to separate the native phosphorylated and nonphosphorylated isoforms of SuSy previously purified from tomato fruit. MATERIALS AND METHODS Materials. All biochemicals were purchased from Sigma (St. Louis, MO). [g- 32P]ATP was from New England Nuclear (Boston, MA), Immobilon-P membranes were from Millipore Ltd. (Bedford, MA), and TLE/TLC cellulose-precoated plates (20 3 20 cm) were from Merck (Darmstadt, Germany). Polyclonal antibodies against maize SuSy were previously obtained and characterized (3). Plant growth conditions. Tomato plants (Lycopersicon esculentum, Mill, cv. UC82B) were grown under greenhouse conditions. Day and night temperatures were maintained at a minimum of 22 and 17°C, respectively. Supplemental lighting (150 mol m 22 s 21 PAR) was

supplied by high-pressure sodium lamps for a 16 h/8 h (light:dark) photoperiod. Extraction, in vitro phosphorylation, and enzyme assay of tomato fruit SuSy. All steps were carried out at 4°C. Twenty-five-day-old tomato fruits were homogenized in ice-cold buffer A (3 ml/g FW) composed of 100 mM Hepes-NaOH (pH 7.5), 3 mM AcMg, 2 mM EDTA, 1 mM PMSF, 4 mM DTT, 0.08% (v/v) 2-mercaptoethanol, 0.1% (v/v) Triton X-100, 10% (v/v) ethylene glycol, 4% (w/v) polyvinylpolypyrrolidone, and 1% (w/v) Dowex-1, chloride form. The homogenate was filtered through a nylon cloth (Spectra/Mesh) and centrifuged at 35,000g for 30 min. In vitro phosphorylation of SuSy by endogenous kinase activity was performed with crude extracts desalted on Sephadex G-25 preequilibrated with a 50 mM HepesNaOH (pH 7.5) buffer, containing 10 mM MgCl 2 and 2 mM DTT (buffer B). The extracts were incubated for 30 min at 28°C in the presence of 10 mM CaCl 2, 0.2 mM ATP, and 25 mCi/ml [g- 32P]ATP. The reaction was stopped by adding EDTA in excess. SuSy activity was assayed in buffer B in the sucrose breakdown direction, at 28°C, in the presence of 250 mM sucrose, using an enzyme-coupling method (23). Tryptic phosphopeptide analysis. In vitro 32P-labeled tomato SuSy was immunoprecipitated, separated by SDS–PAGE (24), and transferred onto an Immobilon-P membrane. Membrane slices containing the ;90-kDa SuSy polypeptide were excised, washed three times in water, saturated for 1 h at 37°C in 100 mM acetic acid, 0.5% (w/v) polyvinylpyrrolidone-40, washed five times with water, and washed twice with digestion buffer [50 mM ammonium bicarbonate, 5% (v/v) acetonitrile]. An aliquot of L-l-p-tosylamino-2-phenylethyl chloromethyl ketone-treated trypsin (10 mg) was added, and the digestion was performed at 37°C for 20 h. An additional 10 mg of trypsin was added after 12 h. The tryptic digest was adjusted at pH 3.0 and the phosphopeptides were semipurified by Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC], as described previously (25). The radioactive fractions were pooled and freezedried. The phosphopeptides were solubilized in 6 mL of pH 1.9 electrophoresis buffer [1% (v/v) formic acid, 10% (v/v) acetic acid] and separated by a two-dimensional TLE/TLC (26). Electrophoresis in the first dimension was performed at 1000 V (20 mA) for 25 min in a Model 1415 electrophoresis cell (Bio-Rad) and in the second dimension for 7 h in a standard chromatographic buffer [40% (v/v) nbutanol, 30% (v/v) pyridine, 6% (v/v) acetic acid]. Phosphoamino acid analysis. The ;90-kDa 32P-labeled SuSy polypeptide immobilized on membrane (see above) was acid hydrolyzed and submitted to a two-dimensional electrophoresis (TLE/ TLE), according to Van der Geer et al. (26). Autoradiography was carried out at 280°C with Kodak BioMax films, using an intensifying screen. Peptide sequencing. Tryptic phosphopeptides from 200 mg of in vitro 32P-labeled tomato fruit SuSy were prepared as described above, except that freeze-drying was stopped when the volume was 500 mL. The sample was brought to 0.1% (v/v) trifluoroacetic acid (TFA) and adjusted to pH 3.0 with 6 N HCl. An aliqut (250 mL) of the resulting mixture was applied to a C 18 reverse-phase HPLC column (3.9 3 300 mm, Nova-Pack) equilibrated in 0.1% (v/v) TFA. The column was developed using a linear acetonitrile gradient (0.8 ml/ min, 1%/min) in 0.08% (v/v) TFA, and fractions of 400 mL were collected and Cerenkov counted. The radioactive fractions were subjected to N-terminus sequencing by automatic Edman degradation performed on an Applied Biosystems Model 473A pulsed liquid protein sequencer. Analysis was performed by the Service de Se´quence de Peptides de l’Est du Que´bec (CHUQ, Sainte-Foy, Que´bec, Canada). Purification of tomato fruit SuSy. Tomato SuSy was purified to homogeneity from 25-day-old tomato fruits using the three-step method described by Nguyen-Quoc et al. (3), with slight modifications. Briefly, proteins precipitated from the crude extract by (NH 4) 2SO 4 (30 –70% saturation) were solubilized in 50 mM Hepes-

PHOSPHORYLATED ISOFORMS OF TOMATO SUCROSE SYNTHASE NaOH (pH 7.5), containing 10 mM MgCl 2, 2mM DTT, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF (buffer C) supplemented with 0.5 mM microcystin LR and dialyzed overnight at 4°C against the same buffer. For in vitro 32P-radiolabeled SuSy, nonincorporated [g- 32P]ATP was removed by desalting on a G-25 sephadex column equilibrated with buffer C, prior to the subsequent purification steps. The sample was loaded onto an UDP-glucuronic acid-agarose affinity column (Sigma). The column was washed with buffer C supplemented with 0.01% (v/v) Triton-X100, and the proteins were then eluted with 0.5 M KCl in buffer C. The fractions containing SuSy activity were pooled, diluted to 100 mM KCl with buffer C, and applied directly to a Mono-Q FPLC column (Pharmacia). SuSy was eluted with a linear gradient of KCl in buffer C. The fractions containing SuSy activity were desalted on Sephadex G-25 equilibrated with buffer C. Homogeneity of purified SuSy was controlled by SDS–PAGE, and the protein sample was brought to 10% (v/v) glycerol and stored at 280°C. Separation of phosphorylated and nonphosphorylated native SuSy isoforms by IMAC. Prepacked Fe(III)-IDA agarose columns (Sigma) were thoroughly washed with water and prepared as previously described (27), except that the columns were finally equilibrated with 50 mM Mes-NaOH (pH 6.3). Purified tomato fruit SuSy was desalted on a G-25 Sephadex column equilibrated with 200 mM Mes-NaOH (pH 6.3) and immediately loaded onto the equilibrated Fe(III)-IDA agarose column. Nonadsorbed SuSy was recovered with the washing buffer (50 mM Mes-NaOH, pH 6.3), and SuSy activity (10 ml/min) was eluted using an increasing stepwise gradient of 0.2 pH units (pH 6.3 to pH 9.5) of a precooled 100 mM Mes-Tris-NaOH buffer. A marker consisting of an aliquot of in vitro 32P-labeled tomato fruit SuSy purified to homogeneity and prepared for Fe(III)IMAC as described above, was then added to the sample prior to Fe(III)-IMAC. SuSy isoforms adsorbed on IDA-agarose were eluted at 4°C with 100 mM Hepes-NaOH (pH 7.5) and 100 mM Tris-NaOH (pH 9.5). The fractions containing SuSy activity were immediately adjusted to 10 mM MgCl 2 and 2 mM DTT and used for the determination of kinetic parameters and peptide mapping. The different buffers used during the experiments were controlled to detect any eventual effect on the stability and the kinetic parameters of the enzyme.

165

FIG. 1. In vitro 32P labeling of soluble proteins from 25-day-old tomato fruit with endogenous protein kinase activity. (A) After labeling in the presence of 0.1 mM Ca 21 or 1 mM EGTA, the proteins (40 mg) were precipitated with 2% trichloracetic acid (a) or immunoprecipitated with maize SuSy-IgGs (b) prior to SDS–PAGE and autoradiography. (B) Effect of Ca 21 and Mg 21 on SuSy phosphorylation was tested in the presence of 4 mM MgCl 2 or 0.1 mM CaCl 2, respectively.

RESULTS AND DISCUSSION

To examine whether tomato SuSy was phosphorylated by an endogenous protein kinase, soluble proteins of 25-day-old tomato fruits were desalted and incubated with [g- 32P]ATP in the presence of calcium or EGTA. As shown in Fig. 1A, the ;90-kDa SuSy subunit incorporated 32P to a relatively high level compared to other soluble proteins, but only in the presence of calcium. The 32P labeling of tomato SuSy was optimal at micromolar calcium concentrations (1–100 mM CaCl 2) and was strongly activated by high magnesium concentrations (up to 10 mM MgCl 2) (Fig. 1B). A requirement of purified plant CDPKs for Mg 21 higher than the concentration needed for the formation of a stable Mg 21–ATP complex has been previously reported (28, 29), with a Mg 21 optimum of 3–10 mM and an inhibitory effect at higher concentrations. The unusual stimulation of in vitro 32P incorporation observed at high Mg 21 (Fig. 1B) may result from conformational modifications of SuSy, a cation-sensitive enzyme (30), or from stabilization of the SuSy/kinase complex. In vitro phosphorylation of soluble and membrane proteins has been studied at different stages of tomato

fruit development (31), where several soluble proteins, including a 90-kDa polypeptide, were phosphorylated by Ca 21-promoted/calmodulin-independent protein kinase activities that generally decreased toward fruit ripening. Although these phosphorylated proteins could potentially be involved in the control of fruit development (31), their identity and respective functions remained unknown. To our knowledge, SuSy is the first phosphorylated protein to be clearly identified in this organ. The drastic effect of EGTA on the phosphorylation of tomato SuSy (Fig. 1A) strongly suggests that SuSy is the substrate of a CDPK in tomato, as in the case of maize leaves (15, 18) and soybean nodules (16). Specific features of the major phosphorylation site identified in maize SuSy (a basic residue in P–3 of the phosphorylated residue and a hydrophobic residue in P–5, P0 being the phosphorylated residue) have been proposed to be responsible for recognition by the SuSy-CDPK (15). Consequently, only one peptidic sequence deduced from TOMSSF (-Leu-Ser-Arg-Ile-Glu-Ser 36-His-Gly-) was a potential substrate for a SuSy-CDPK. Thus,

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FIG. 2. Amino acid mapping (A) and tryptic peptide mapping (B) of in vitro 32P-labeled tomato fruit SuSy. After immunoprecipitation, SDS–PAGE, and transfer to Immobilon-P, the radiolabeled 90-kDa polypeptide was excised and used for amino acid and peptide mapping analyses. P-ser, phosphoserine; P-thr, phosphothreonine; P-tyr, phosphotyrosine. The origins are marked with a cross.

experiments were carried out to identify the phosphorylation site(s) of tomato SuSy targeted by the endogenous protein kinase(s). First, in vitro 32P-labeled tomato SuSy was immunopurified, submitted to SDS– PAGE, and transferred onto an Immobilon-P membrane (see Materials and Methods). Amino acid mapping of the immobilized 90-kDa SuSy polypeptide indicated that tomato SuSy was phosphorylated only on seryl residue(s) (Fig. 2A). Two-dimensional peptide mapping (TLE/TLC) of the tryptic digest (Fig. 2B) resolved a major 32P-labeled peptide (named Ppt1) relatively mobile in the electrophoretic dimension and at least one additional, less mobile 32P-labeled peptide (named Ppt2). These results suggest that two distinct seryl residues of tomato SuSy could be phosphorylated in vitro by an endogenous CDPK activity. However, care must be taken when interpreting tryptic peptide analysis as partial tryptic digestion or chemical modifications may produce artifactual spots on the peptide map (32). In a separate experiment, the tryptic digest of in vitro 32P-labeled SuSy of 25-day-old tomato fruit has

been submitted to RP-HPLC (Novapack) analysis. As shown in Fig. 3A, about 90% of the incorporated radioactivity was eluted in 13.5% acetonitrile (peak A), and a lower peak was resolved in 18.5% acetonitrile (peak B). Peptide mapping of peak A resolved a single 32Ppeptide, which corresponded to Ppt1 by its position after TLE/TLC (Fig. 3B). The amino acid sequence of this tryptic phosphopeptide was -Val-His-(X)-Leu-Arg(Table I). The presence of a phosphoseryl residue in the third position of Ppt1 was indicated by the production of phenylthiodantoin– dehydroalanine (X), a dithiothreitol adduct formed by addition of a thiol to the b-elimination products of phosphoserine or phosphothreonine. Phosphoamino acid analysis of Ppt1 recovered from the TLC-cellulose plate confirmed the presence of a phosphoserine residue in this peptide (Fig. 3C). The peptide sequence of Ppt1 corresponded to the major phosphorylation site of maize SuSy identified by Huber et al. (15), and was also identical to other dicotyledonous SuSy, including the potato SuSy isoforms Sus3 and Sus4 (9). To verify that the presence of an arginine residue at position 11 of the TOMSSF polypeptide (21) was not a sequencing artifact, the region between nucleotides 5 and 500 of TOMSSF was amplified by RT-PCR from tomato fruit mRNAs and cloned into a plasmid vector. The nucleotide sequence of a selected clone confirmed the presence of an arginine residue in position 11 of the deduced TOMSSF polypeptide, indicating that a SuSy isozyme differing from the TOMSSF polypeptide by at least one seryl residue in the N-terminus extremity was expressed in tomato fruit. The relatively high incorporation of 32P into tomato fruit SuSy, attributed mainly to the phosphorylation of the seryl residue described above, suggested that the corresponding SuSy isozyme is a major form in this organ. The 32P-peptide eluted in peak B by RP-HPLC (Fig. 3A) was also resolved by TLE/TLC and identified as Ppt2 according to its localization on the chromatographic plate (Fig. 3B). Unfortunately, there was insufficient Ppt2 for sequencing. Thus, further analysis is necessary to determine the sequence of this phosphopeptide. To determine the effect of phosphorylation on the kinetics of tomato SuSy, experiments were conducted to obtain purified nonphosphorylated and phosphorylated tomato fruit SuSy. The treatment of 32P-labeled tomato SuSy with alkaline phosphatase (CIP) or potato acid phosphatase led to a poor removal of radiolabeling from the protein in its native form (data not shown). Therefore, we used Fe(III)-IMAC to isolate the phosphorylated fraction of purified tomato fruit SuSy. Fe(III)-IMAC has been used previously for the separation of phosphorylated biomolecules on the basis of interactions with hard Lewis bases such as phosphate (33, 34) and was adapted here for a native plant enzyme. To avoid nonspecific binding to the matrix, the sample was equilibrated at pH 6.3, which is slightly

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PHOSPHORYLATED ISOFORMS OF TOMATO SUCROSE SYNTHASE

forms that could be resolved from the affinity column (see Materials and Methods). Three peaks of SuSy activity were eluted at pH 6.3 (nonadsorbed), pH 6.7, and pH 8.9 (data not shown). Then, an aliquot of purified in vitro 32P-labeled tomato SuSy (7000 CPM) was added to the sample and the elution was performed with a three-step pH gradient (Fig. 4A). A nonradiolabeled fraction (45% of the loaded SuSy activity) was not adsorbed on Fe(III)-IDA-agarose (peak I), while two radiolabeled peaks containing 40% (peak II) and 15% (peak III) of the loaded SuSy activity were resolved at pH 7.5 and pH 9.5, respectively. The elution pattern was also similar in the presence of 0.8 M KCl (data not shown), confirming that phosphate content rather than electrostatic interactions was responsible for the specific interaction of the enzyme with the affinity column. As the radioactive marker represented only 5% of the loaded sample, the relative ratio between the nonphosphorylated (peak I) and the phosphorylated forms (peaks II and III) was likely representative of the phosphorylation status of tomato SuSy in vivo. Since the strength of binding depends on the phosphate content of the protein (27, 33), the separation of two radiolabeled SuSy fractions by Fe(III)-IMAC might reflect the presence of two phosphorylated tomato SuSy isoforms differing by the number of phosphate incorporated into the tetrameric protein. The higher phosphate content of SuSy from peak III was indicated first by the stoichiometry of 32P incorporation ( 32P radioactivity per unit of SuSy activity), which was about twofold higher in peak III than in peak II (Fig. 4A), and second by the tryptic peptide maps of peak II and peak III (Fig. 4B). A single 32P-labeled peptide (Ppt1, by analogy with the map presented in Fig. 2B) was observed in the peptide map of peak II, indicating that this tomato Susy isoform was phosphorylated only at the seryl residue related to the major phosphorylation site of maize SuSy (Fig. 4B). The peptide map of peak III (Fig. 4B) was similar to the map presented in Fig. 2B, except that both radioactive spots (Ppt1 and Ppt2) had a similar intensity of 32P radioactivity. As tryptic digestions of TABLE I

Automatic Edman Degradation of the Tryptic 32P-Labeled Phosphopeptide PPt1 after Purification by Fe(III)-IMAC and RP-HPLC (Novapack) FIG. 3. RP-HPLC (Novapack) of the tryptic digest of in vitro 32Plabeled tomato fruit SuSy (A). Fractions of 400 mL were collected. Aliquots (500 CPM) of peaks A and B were lyophilized and resolved by two-dimensional TLE/TLC (B). Ppt1 was recovered from the cellulose plate and submitted to phosphoamino acid analysis, except that TLE was performed in one dimension at pH 3.5 (C). P-S, phosphoserine; P-T, phosphothreonine; P-Y, phosphotyrosine. The origins are marked with a cross.

above the isoelectric points commonly reported for SuSy (GenBank). An increasing stepwise pH gradient was first used to determine the number of SuSy iso-

Cycle

Amino acid

Amino acid in deduced sequence a

Position in deduced sequence

1 2 3 4 5

VAL HIS Xb LEU ARG

VAL HIS ARG LEU ARG

9 10 11 12 13

a b

From the nucleotide sequence of the tomato cDNA TOMSSF. A phenylthiodantoin– dehydroalanine peak was observed.

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FIG. 4. Elution profiles of SuSy activity (h) and radioactivity (F) by Fe(III)-IMAC (A). SuSy was purified to homogeneity from tomato fruit and equilibrated in Mes-NaOH 200 mM (pH 6.3) prior to metal affinity chromatography. An aliquot of radiolabeled SuSy (7000 CPM, 5% of SuSy activity loaded) was used as a marker. Fractions of 500 mL were recovered and elution buffers were changed as indicated by arrows: x, Mes-NaOH 50 mM (pH 6.3); y, Hepes-NaOH 100 mM (pH 7.5); z, Tris-NaOH 100 mM (pH 9.5). (Inset) The sample applied to the affinity column was controlled by SDS–PAGE and autoradiography. Aliquots of peaks II and III were immunoprecipitated with maize SuSy-IgGs and analyzed by tryptic peptide mapping (B).

peak II and peak III fractions were carried out in parallel with the same trypsin preparation, the single spot observed in the tryptic map of peak II (Fig. 4B) provided evidence that Ppt2 was not produced by chemical modification or incomplete tryptic digestion of Ppt1, but instead reflected the presence of an addi-

tional phosphorylation site. Taken together, these results strongly suggest that phosphorylation of the tetrameric SuSy protein at two different phosphorylation sites was responsible for the higher affinity of the peak III fraction to Fe(III)-IDA. Moreover, phosphorylation of the major phosphorylation site of tomato SuSy could be a prerequisite for the subsequent phosphorylation of a second phosphorylation site. Kinetic parameters of the tomato SuSy isoforms purified by Fe(III)-IMAC were examined in the sucrose cleavage direction. As shown in Table II, specific SuSy activities and K m for sucrose measured in the different samples were almost identical. However, a marked decrease of the K m for UDP was observed in the peak III fraction compared to the nonphosphorylated fraction (peak I), while this parameter remained unchanged for the peak II fraction. Therefore, multiphosphorylation of SuSy may promote sucrose cleavage in tomato fruit by increasing its affinity for UDP. As serine 15 represented the major if not the only in vivo phosphorylation site detected in maize shoots (15, 18), the presence of additional phosphorylation sites of SuSy should be tested in other plant tissues. The recent report 3 showing that PKA is able to phosphorylate a recombinant form of soybean SuSy lacking the Nterminus phosphorylation site is consistent with the presence of other potential phosphorylation site(s) on SuSy. Our results also clearly demonstrate that phosphorylation of the major site of tomato SuSy does not affect by itself the sucrose cleavage properties of the enzyme. As shown by Winter et al. (17), phosphorylation/dephosphorylation of this N-terminus site may rather represent an important mechanism controlling the cellular localization of maize SuSy by increasing its surface hydrophobicity. Unlike partially dephosphorylated maize SuSy that tends to form a precipitate when kept at 4°C (17), the activity of the nonphosphorylated tomato SuSy isoform (peak I) was not affected after a 3 Zhang, X-Q., and Chollet, R. (1998) Annual Meeting of the A.S.P.P., Abstract No. 534 (Madison, WI).

TABLE II

Kinetic Parameters of Purified Tomato Fruit SuSy Isoforms in the Sucrose Cleavage Direction Parameter

SuSy a

Peak I b

Peak II

Peak III

Activity (UI/mg Prot.) Residual activity (%) c K m UDP (mM) d K m SUC (mM) e

14.78 6 1.52 96.25 6 5.32 25.25 6 3.12 71.63 6 4.45

14.50 6 0.73 98.16 6 4.54 31.38 6 4.17 64.81 6 1.40

15.31 6 0.88 92.40 6 2.14 27.21 6 4.12 62.96 6 5.07

14.55 6 0.70 97.17 6 1.88 10.30 6 1.95 57.89 6 1.52

Data are means 6 SE of four separated purifications of SuSy from 25-day-old tomato fruit. Purified SuSy was desalted in 200 mM Mes-NaOH (pH 6.3) and separated in three fractions (peak I, peak II, and peak III) by Fe(III)-IMAC. Data are means 6 SE of three measurements. c After 48 h incubation at 4°C in buffer B. d Measurement with 250 mM sucrose. e Measurement with 100 mM UDP. a b

PHOSPHORYLATED ISOFORMS OF TOMATO SUCROSE SYNTHASE

48-h incubation at 4°C (Table II). However, it should be noted that detergent extraction procedures used in our study may have facilitated the solubilization of membrane-bound SuSy. Since the activity of membrane SuSy could provide an appreciable source of UDP-Glc for cell wall polysaccharide biosynthesis (35–37), factors that control the localization of SuSy in tomato fruit cells may be critical for fruit growth and/or in response to a pathogen attack. Thus, possible implication of the N-terminus phosphorylation site of tomato SuSy isozymes will need to be addressed in the future. To our knowledge, the present study reports for the first time the separation of isoforms of native enzymes by immobilized metal affinity chromatography based on their phosphate content, without loss of biological activity. In our opinion, Fe(III)-IMAC will be useful for the purification of many other phosphoenzymes and could be an alternative to in vivo 32P labeling, which requires the manipulation of large amounts of 32P, a rapid turnover of protein-bound phosphate, and perturbation of plant integrity. ACKNOWLEDGMENTS We thank Dr. Dominique Michaud for helpful comments on the manuscript. This work was supported in part by grants from the Natural Science and Engineering Research Council of Canada to S.Y.

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