©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Immunological Characterization and Chloroplast Localization of the Tryptophan Biosynthetic Enzymes of the Flowering Plant Arabidopsis thaliana(*)

(Received for publication, December 8, 1994; and in revised form, December 23, 1994)

Jianmin Zhao Robert L. Last

From the Boyce Thompson Institute for Plant Research and Section of Genetics and Development, Cornell University, Ithaca, New York 14853-1801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.


INTRODUCTION

The study of the tryptophan biosynthetic pathway in bacteria and fungi has contributed significantly to our understanding of microbial genetic regulation and biochemistry(1, 2) . Plants appear to have the same tryptophan biosynthetic pathway as microorganisms (Fig. 1). However, the tryptophan pathway also provides precursors for the plant hormone auxin and many other indolic secondary products in higher plants(3) . While the pathway is important for plant development and defense, little is known about the regulation of the plant tryptophan biosynthetic enzymes. To address this problem, tools are being developed to allow detailed studies of this pathway in Arabidopsis thaliana. For example, in contrast to the paucity of amino acid auxotrophs in other plant species, Arabidopsis mutants are available for four of the tryptophan biosynthetic pathway enzymes. As shown in Fig. 1, trp4 mutants are defective in anthranilate synthase beta subunit(4) , trp1 mutants lack phosphoribosylanthranilate transferase activity(5, 6) , and trp3 and trp2 mutants have defects in the tryptophan synthase alpha (^1)and beta (7) (^2)subunits, respectively.


Figure 1: Tryptophan biosynthetic pathway in Arabidopsis. InGPS, indole-3-glycerol-phosphate synthase (EC 4.1.1.48). Enzymes against which antibodies were generated are indicated in bold letters. Locus designations for mutants in the pathway are indicated to the left of the arrows (trp1-trp4).



Biochemical studies indicate that the plant aromatic amino acid biosynthetic pathways are localized to the chloroplast(8, 9) . Consistent with these results, protein precursors of plant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and 5-enol-pyruvylshikimate-3-phosphate synthase, the first and penultimate enzymes in the shikimate pathway, can be imported into chloroplasts in vitro(10, 11) . Protein sequences deduced from cDNA sequences of all cloned tryptophan biosynthetic enzymes from Arabidopsis contain putative chloroplast-targeting peptides at their NH(2) termini(3, 4, 6, 7, 12, 13) . (^3)(^4)To our knowledge there have been no reports showing that the precursors encoded by the cDNAs are imported into chloroplasts. Isolated Arabidopsis chloroplasts were shown to contain more than 98% of the tryptophan synthase beta activity(7) . We are unaware of any other direct biochemical demonstration that tryptophan biosynthetic enzymes are present in chloroplasts.

In order to characterize the wild-type and mutant tryptophan biosynthetic proteins and to explore the regulation of protein product accumulation during development and in response to environmental factors, we have produced polyclonal antibodies against anthranilate synthase alpha subunit (ASA), (^5)phosphoribosylanthranilate transferase (PAT), phosphoribosylanthranilate isomerase (PAI), as well as the tryptophan synthase alpha (TSA) and beta subunits (TSB) from Arabidopsis. These antibodies were used to show that at least 90% of the ASA, PAT, PAI, TSA, and TSB proteins from Arabidopsis leaves were localized in the chloroplasts. To test whether the cloned cDNAs of tryptophan biosynthetic enzymes encode chloroplast-localized proteins, PAI and TSA cDNAs were used to synthesize PAI and TSA protein precursors in vitro. These precursors were shown to be imported into and processed by isolated spinach chloroplasts. NH(2)-terminal sequencing of the processed proteins revealed the location of the cleavage sites.


EXPERIMENTAL PROCEDURES

Materials and General Procedures

Escherichia coli strain DH5alpha [supE44 DeltalacU169 (80 lacZ DeltaM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used as a bacterial host unless otherwise indicated. Alkaline phosphatase-conjugated goat anti-rabbit antibody and protein molecular weight standards were from Bio-Rad. In vitro transcription kit, RNasin, T7 RNA polymerase, and rabbit reticulocyte lysate were from Promega. [S]Methionine, [^3H]Leucine, and I-protein A were purchased from DuPont NEN. Nitrocellulose (0.2 µm) was purchased from Schleicher and Schuell. Standard DNA and protein procedures were performed as in (14) , unless stated otherwise.

Growth Conditions

The mutant trp1-3 (mutation in PAT1 gene) (^6)and its parent A. thaliana Landsberg erecta (Ler) were grown on PNS medium (15) supplemented with 50 µM tryptophan for 3 weeks under constant illumination (80 microeinsteins). A. thaliana Columbia (Col-0) and mutants trp2-8 (mutation in TSB1 gene)^2 and trp3-1 (mutation in TSA1 gene)^1 were grown in soil under constant light (60-80 microeinsteins) at 21-23 °C. E. coli strains were grown in LB medium at 30 or 37 °C. Spinach was grown on soil in a greenhouse under a 16-h light and 8-h dark photoperiod.

Construction of Plasmids for the Expression of GST Fusion Proteins

pGEX1 and pGEX2T (Pharmacia Biotech Inc.) (16) were digested with SmaI and treated with alkaline phosphatase. The ClaI-KpnI fragment from PAT1 cDNA pAR129(6) , the PstI-PstI fragment from PAI2 cDNA pJL24,^4 and the NciI-EcoRI fragment from TSB1 cDNA pCD7B (17) were rendered blunt-ended with Klenow fragment and ligated into the SmaI site of pGEX1 to create pGEX-PAT, pGEX-PAI, and pGEX-TSB, respectively. The NcoI-EcoRI fragment from TSA1 cDNA pERR65 was blunt-ended with Klenow fragment and ligated into the SmaI site of pGEX2T to create pGEX-TSA.^1 This filling-in by DNA polymerase during the construction of pGST-PAT and pGST-PAI created codons that encode proline and arginine, respectively, that are not encoded by either the vector or the cDNA inserts.

Purification of Fusion Proteins

GST fusion proteins were purified by a published method (16) with glutathione-agarose beads (Sigma). The eluted fusion proteins were further purified by preparative electrophoresis on 10% SDS-polyacrylamide gels. These gels were stained with 0.05% Coomassie Brilliant Blue (in water) for 10 min and destained in water until the protein bands became apparent. The fusion protein bands were excised from the gel and electroeluted in a dialysis bag with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) running buffer at 100 V for 3 h at 4 °C.

Production of Antibodies

300 µg of the purified GST-ASA (kindly provided by Paul Bernasconi and Mani Subramanian of Sandoz Agro, Inc.)(18) , GST-TSA, and GST-TSB and 100 µg of purified GST-PAT and GST-PAI were emulsified with Freund's complete adjuvant and injected subcutaneously into 3-kg rabbits (Flemifh Giant-Chinchilla). For booster injections, the same mass of protein as in the initial injection was emulsified into Freund's incomplete adjuvant and injected subcutaneously. Boosters were given four times for GST-ASA, GST-TSA, and GST-TSB and twice for GST-PAT and GST-PAI at 2-3-week intervals. The blood from these rabbits was collected 3 weeks after the last injection. The blood was then incubated at 37 °C for 1 h and at 4 °C overnight, and the serum was obtained by centrifugation at 5,000 times g for 15 min at 4 °C.

Affinity Purification of the Antibodies

Glutathione agarose affinity-purified GST-PAT, GST-PAI, GST-TSA, or GST-TSB fusion protein was bound to nitrocellulose membranes by filtration of the protein solutions under vacuum. The membranes were then incubated overnight at 4 °C with a 1:200 dilution of antiserum and washed three times with TBS (25 mM TrisbulletHCl, 0.14 M NaCl, pH 7.4). Antibodies were eluted from the washed membranes with 1 ml of 100 mM glycine, pH 2.0, and the eluate was immediately neutralized with 1/8 volume of 2 M TrisbulletHCl, pH 8.0.

Protein Sample Preparation and SDS-PAGE

SDS-PAGE protein samples from E. coli and glutathione affinity chromatography were prepared as described previously(14, 16) . Arabidopsis protein samples were prepared by homogenizing 50 mg of Arabidopsis rosette leaf tissue with 200 µl of extraction buffer (100 mM KPO(4), 0.14 M NaCl, 1 mM phenylmethylsulfonyl fluoride, pH 7.4). The resulting homogenates were spun for 5 min in a microcentrifuge at 16,000 times g, and the supernatants were transferred to new tubes and kept on ice. Protein concentrations of these samples were determined by the Bradford method(19) . Protein concentrations were adjusted with extraction buffer prior to SDS-PAGE. For the chloroplast import assays, SDS-PAGE samples were prepared by adding 1 times SDS sample buffer directly to the various samples.

All protein samples were heated at 95-100 °C for 4 min and loaded onto a 12% SDS-polyacrylamide gel, using the Mini-PROTEAN II gel system (Bio-Rad). The gels were run at 120 V for 75 min at room temperature. For the import studies, the gels were fixed, stained with Coomassie Blue, destained, and dried under vacuum on 0.35-mm filter paper (Fisher) at 80 °C. The dried gels were quantified using a PhosphorImager (Molecular Dynamics) or subjected to autoradiography with Kodak X-OMAT film.

Immunoblots

The proteins from SDS-PAGE gels were transferred to nitrocellulose at 35 V overnight with a Mini Trans-Blot system (Bio-Rad). The nitrocellulose membranes were stained with Ponceau S (Sigma) for detection of total proteins and subsequently blocked with Blotto (5% nonfat dry milk, 25 mM TrisbulletHCl, 0.14 M NaCl, pH 7.4) for 4-16 h at room temperature. The membranes were then incubated for 4 h at room temperature or overnight at 4 °C with antibodies diluted in Blotto. Anti-ASA serum was used at 1:1000 dilution, and affinity-purified anti-PAT, -PAI, -TSA, and -TSB antibodies were used at 1:250, 1:100, 1:1,000, and 1:1,000 dilutions, respectively. The membranes were then washed three times with Blotto and incubated with either alkaline phosphatase-conjugated goat anti-rabbit antibody or I-protein A. Visualization was by color reaction with 5-bromo-4-chloro-3-indolyl phosphate-toluidine salt and p-nitro blue tetrazolium chloride for alkaline phosphatase-conjugated goat anti-rabbit antibody or autoradiography with the PhosphorImager or Kodak X-OMAT film for I-protein A.

In Vitro Transcription and Translation of PAI and TSA

Plasmids for in vitro transcription of PAI2 (pJL167)^4 and TSA1 (pERR65) were linearized at the 3` end of the inserts with XbaI and SmaI, respectively. The linearized plasmids were used as templates for the synthesis of uncapped RNA by T7 RNA polymerase. In vitro translations were carried out in the presence of [S]methionine and [^3H]leucine with rabbit reticulocyte lysate as described by the supplier (Promega). The in vitro translation products were either used immediately for import experiments or stored at -70 °C.

Uptake of PAI and TSA Precursors by Isolated Spinach Chloroplasts and Isolation of Arabidopsis Chloroplasts

Isolation of intact chloroplasts from spinach and Arabidopsis leaves, chlorophyll measurement, import assays of PAI and TSA precursors into spinach chloroplasts, treatment of the import reaction medium by trypsin and chymotrypsin, and chloroplast subfractionation were carried out as described(20) . Spinach chloroplasts were employed rather than those from Arabidopsis because published methods allow these to be obtained in adequate quantity and purity.

Radioactive Sequencing of PAI and TSA

PAI and TSA precursors were imported into isolated spinach chloroplasts as described above. Chloroplasts were reisolated, and the soluble protein fraction was obtained as described(20) . The proteins in the soluble fraction were precipitated with 10% trichloroacetic acid, washed twice with ethyl acetate, boiled in SDS sample buffer, and subjected to SDS-PAGE. Proteins from the gel were transferred to an Immobilon-P membrane (Millipore), and the labeled mature proteins were visualized by autoradiography with Kodak X-OMAT film. The portions of the membrane containing processed PAI and TSA proteins were excised and subjected to automatic Edman degradation. The samples from each cycle were collected, dried, resuspended in scintillation fluid, and counted with a Beckman LS7500 scintillation counter.


RESULTS

Overexpression and Purification of Fusion Proteins

In order to obtain enough protein of adequate purity to make antibodies, plasmids were constructed to overexpress GST-PAT, -PAI, -TSA, and -TSB fusion proteins in E. coli. The putative transit peptides of these proteins were not included in the fusion proteins because they are unlikely to be present in the mature proteins. We predicted that the NH(2) termini for the mature proteins would be close to the residues where the plant sequences start to show similarities with the microbial sequences. Convenient restriction sites close to these predicted NH(2) termini were used to construct the expression plasmids. The expression plasmids encode fusion proteins with the GST moiety translationally fused to the Arabidopsis PAT, PAI, TSA, or TSB enzymes lacking the NH(2)-terminal 55, 45, 37, or 68 amino acids from their cDNA deduced protein sequences, respectively (Fig. 2A). The desired constructs were identified by restriction enzyme analysis of plasmid DNAs followed by analysis of the GST fusion proteins by SDS-PAGE. The fusion proteins purified with glutathione-agarose beads had apparent molecular masses of 72 (GST-PAT), 49 (GST-PAI), 56 (GST-TSA), and 65 kDa (GST-TSB), as determined by SDS-PAGE (Fig. 2B). These values agree reasonably well with the calculated masses of these fusion proteins (69, 52, 57, and 72 kDa, respectively). To ensure the purity of the proteins used for antibody production, the affinity-purified fusion proteins were subjected to preparative SDS-PAGE. Fig. 2B shows that this procedure resulted in single protein species as judged by Coomassie Blue staining. The GST-ASA fusion protein was provided by Paul Bernasconi and Mani Subramanian(18) .


Figure 2: Fusion proteins between glutathione S-transferase and the Arabidopsis tryptophan biosynthetic proteins. A, the inferred amino-terminal sequences of the Arabidopsis PAT, PAI, TSA, and TSB proteins. The arrowheads indicate the first amino acid included in each GST fusion protein, while the asterisks mark the amino termini of the mature PAI and TSA proteins. The 27.5-kDa GST protein is fused to truncated PAT, PAI, TSA, and TSB coding regions that have calculated molecular masses of 40.8, 24.5, 29.1, and 44.0 kDa, respectively. The coding region sequences are derived from DNA sequences (PAT: (6) , GenBank Accession No. M96073B; PAI: J. Li et al., submitted for publication, GenBank Accession No. U18968; TSA: E. R. Radwanski et al., manuscript in preparation, GenBank Accession No. U18993; TSB: (7) , GenBank Accession No. M23872). B, Coomassie Blue-stained SDS-PAGE of the GST-PAT, GST-PAI, GST-TSA, and GST-TSB fusion proteins purified by glutathione-agarose affinity chromatography and preparative gel electrophoresis. Molecular masses calculated based upon comparison with known size standards are indicated.



Production and Characterization of the Antibodies

Polyclonal antibodies against the fusion proteins GST-ASA, -PAT, -PAI, -TSA, and -TSB were raised in rabbits. The GST-PAT, -PAI, -TSA, and -TSB fusion proteins were used to purify their corresponding antibodies. These affinity-purified antibodies were used to demonstrate that the antibodies identified proteins of the expected molecular weights in Arabidopsis leaf extracts. As Fig. 3shows, antibodies against PAT, PAI, TSA, and TSB recognized proteins with apparent molecular masses of 40, 31, 30.5, and 42 kDa, respectively (lanes labeled Arabidopsis). To test the specificity of the antibodies, E. coli strains expressing cDNAs encoding Arabidopsis PAT, PAI, TSA, and TSB were analyzed by immunoblots. The antibodies recognized proteins unique to E. coli strains expressing the cognate cDNA (Fig. 3, A-D, first three lanes of each panel). Protein bands present in all three E. coli strains represent nonspecific cross-reacting materials from E. coli. Because these nonspecific bands are not present in Arabidopsis leaf extracts, they are not likely to interfere with the analysis of the Arabidopsis proteins by the antibodies. Proteins of higher molecular weight than those present in the leaf extract were expected for E. coli expressing PAT, PAI, and TSA cDNAs because these clones should produce proteins with putative chloroplast target sequences at the NH(2) termini. In contrast, the Arabidopsis TSB cDNA encodes a truncated form of the TSB precursor with the first 42 amino acid residues missing from its NH(2) terminus. As expected, antibodies against PAI and TSA recognized only protein bands present in E. coli expressing the cognate cDNAs that are larger than the corresponding mature proteins from Arabidopsis (Fig. 3, B and C). However, very little PAT precursor-sized cross-reactive protein was found in the E. coli expressing the PAT cDNA (Fig. 3A). Multiple bands observed in E. coli expressing PAT, PAI, and TSA cDNAs were most likely caused by proteases in E. coli. Some of these proteins have molecular weights similar to those found in Arabidopsis leaf extracts (Fig. 3, A-C). These results demonstrate that the antibodies recognize the corresponding Arabidopsis proteins expressed in E. coli, and the data are consistent with the hypothesis that the PAI and TSA cDNAs encode larger molecular weight precursors.


Figure 3: Antibodies raised against fusion proteins recognize Arabidopsis tryptophan biosynthetic enzymes. Extracts from Arabidopsis leaf or from E. coli expressing Arabidopsis PAT, PAI, or TSA and TSB are as indicated above each lane. The blots were probed with antibodies against PAT (A), PAI (B), TSA (C), and TSB (D). The positions of the mature proteins from Arabidopsis and the protein molecular mass standards are indicated. The calculated masses for unprocessed PAT, PAI, TSA, and TSB proteins are 46,517, 29,617, 33,196, and 50,922, respectively. Protein bands that are present in all three E. coli strains represent nonspecific reaction of the antibodies with E. coli proteins. The blots were visualized by alkaline phosphatase-conjugated goat anti-rabbit antibody with substrates 5-bromo-4-chloro-3-indolyl phosphate-toluidine salt and p-nitro blue tetrazolium chloride.



To further test the specificity of these antibodies, we determined the level of cross-reactive material in Arabidopsis mutants defective in three steps of the tryptophan biosynthetic pathway. We reasoned that the absence or reduction of cross-reactive material in a structural gene mutant would provide strong evidence that the antibody preparation is specific for the desired protein. Fig. 4A shows that protein extract from the PAT activity-deficient mutant trp1-3 is missing the 40-kDa PAT protein present in the parent Arabidopsis Ler. (^7)When anti-TSB was used to probe the TSB mutant trp2-8,^2 the amount of the 42-kDa protein detected in the Col-0 wild type was greatly reduced in extract of the mutant (Fig. 4B). Finally, Fig. 4C shows that the 30.5-kDa protein species detected by anti-TSA in the Col-0 wild type is absent from the TSA mutant trp3-1.^1 Similar amounts of total proteins were loaded in all cases, as shown by staining the membranes with Ponceau S prior to immunological detection (data not shown), and by probing the extracts with the anti-ASA antiserum (Fig. 4D). These results provide convincing evidence that the anti-PAT, -TSA, and -TSB antibodies are specific for the targeted proteins.


Figure 4: Immunoblots of leaf extracts from wild-type and selected Arabidopsis trp mutants developed with anti-PAT (A), anti-TSB (B), anti-TSA (C), and anti-ASA (D). Equal amounts of protein from Arabidopsis leaf were loaded onto each lane. The positions of the mature tryptophan biosynthetic enzymes are indicated on the left side of each blot, whereas the positions of the prestained molecular mass standards are indicated on the right. I-Protein A was used as the secondary detection agent. The blots were visualized by autoradiography.



In Vitro Import and Processing of PAI and TSA Protein Precursors by Isolated Chloroplasts

The tryptophan biosynthetic pathway enzymes are proposed to be in the chloroplasts of plants(8, 9) . Presumably, the higher molecular weight cross-reactive proteins that accumulated in E. coli expressing Arabidopsis PAI and TSA cDNAs (Fig. 3, B and C) were unprocessed precursor proteins. If this were true, these putative precursor proteins should be competent for uptake into chloroplasts and proteolytic processing to produce proteins of the same molecular weight as found in the Arabidopsis extracts. To test this assumption, we produced PAI and TSA precursor proteins in vitro using a rabbit reticulocyte lysate programmed by in vitro transcribed PAI and TSA mRNAs (Fig. 5, A and B, lanes labeled translation). As predicted, the molecular masses of the PAI and TSA in vitro translation products (34 and 37 kDa) were similar to the putative precursors made in E. coli ( Fig. 3and Fig. 5). In Fig. 5, the labeled proteins with molecular masses smaller than those of the precursors in the in vitro translation reactions are likely to result from protein degradation or incomplete translation.


Figure 5: In vitro processing and uptake of the Arabidopsis PAI and TSA precursors into isolated spinach chloroplasts. [S]Methionine- and [^3H]leucine-labeled PAI (A) and TSA (B) protein precursors were synthesized with rabbit reticulocyte lysate using in vitro transcribed RNA (lanes labeled translation). The precursors were incubated with isolated chloroplasts for 20 min at room temperature with shaking to keep the chloroplast suspended. These total import media (lanes labeled import medium) were then incubated on ice with (lanes under protease) or without a mixture of trypsin and chymotrypsin for 30 min. Chloroplasts were reisolated from the import medium and separated into soluble (soluble) and membrane (membrane) fractions by centrifugation. An unlabeled Arabidopsis leaf protein extract was included on each gel. The proteins separated on the gel were transferred to nitrocellulose membrane. The Arabidopsis leaf proteins were detected by immunoblots with I-protein A as the secondary reagent. Pre- and M- indicate the positions of the precursor and processed mature-sized proteins, respectively.



The results of the import studies showed that the precursors of PAI (Fig. 5A) and TSA (Fig. 5B) were imported into chloroplasts and processed into their mature forms. To test whether the mature PAI and TSA proteins were not just adsorbed to but actually taken up by the chloroplasts, protease treatment was carried out after the import assay. The protease digested the precursors and other extraplastidic proteins, while the mature PAI and TSA proteins inside the chloroplasts were protected from degradation (Fig. 5, lanes under protease). Reisolation of chloroplasts after the import reaction revealed that the soluble fraction of the chloroplast contained processed PAI and TSA proteins (Fig. 5, lanes labeled soluble). This result is consistent with a stromal localization for the imported and processed proteins. Although reisolated chloroplast membrane fractions contained precursor proteins (Fig. 5, lanes labeled membrane), these precursors were protease-sensitive (Fig. 5, lanes labeled membrane under protease), indicating that they were not taken up by the chloroplasts.

Time course studies of the import reactions indicated that the accumulation of mature PAI and TSA inside the chloroplasts reached a maximum in about 20 min (Fig. 6, A-D). The in vitro translation products of PAI contained very little mature-sized PAI proteins. Mature-sized PAI started to accumulate immediately following the incubation of the precursors with the chloroplasts (Fig. 6, A and B), indicating that the import of PAI is a rapid process. In contrast to PAI, the strong signal at the position of mature TSA protein in the unprocessed translation mixture (Fig. 6C, inset lane T) made it difficult to assay the TSA import reaction. Analysis of reisolated chloroplasts allowed us to assay directly the accumulation of labeled mature TSA proteins in chloroplasts (Fig. 6D). The decrease in TSA precursor correlates with the accumulation of the mature TSA in the reisolated chloroplasts (Fig. 6, C and D), indicating that these precursors were converted into the mature TSA. These results show that the full-length PAI and TSA translation products are imported into chloroplasts and that the chloroplasts process the precursors into their mature proteins in vitro.


Figure 6: Time course of precursor processing and import into isolated chloroplasts. PAI (A and B) or TSA (C and D) precursors labeled with [S]methionine and [^3H]leucine were incubated with isolated spinach chloroplasts at room temperature with shaking. Samples were taken from the import medium at the indicated time. The conversion of precursors into mature forms was monitored by assaying the import medium (A and C). Chloroplasts were reisolated from the import medium (B and D) to directly monitor the chloroplast-associated proteins. The radioactivities of the precursors and mature proteins separated by SDS-PAGE were quantified by PhosphorImager. The insets show autoradiograms of the SDS-PAGE used in the quantitations with the incubation times indicated. The letter T above the gel indicates the in vitro translation products before incubation with chloroplasts.



Determination of the NH(2) Termini of the Processed PAI and TSA

To define the precise cleavage sites in the PAI and TSA precursors, the NH(2) termini of the in vitro processed PAI and TSA were determined. Fig. 7shows the radioactive sequencing profiles of processed PAI and TSA labeled with [^3H]leucine and [S]methionine. Cycle 21 of PAI is high in ^3H indicating a leucine residue, and cycles 24 and 35 are high in S indicating methionine at these positions (Fig. 7A). The high radioactivity in cycles 22 (^3H) and 25 (S) are likely to be spillover from the previous cycles. Comparing the PAI sequencing data with the deduced PAI protein sequence (Fig. 2A), we predict Ser-46 to be the NH(2)-terminal residue of the processed PAI protein. Cycles 2, 9, and 11 of TSA sequencing are high in ^3H indicating leucine residues at these positions (Fig. 7B). Comparison of these leucine positions with the deduced TSA protein sequence (Fig. 2A) indicates that the TSA cleavage site is between 40 and 41, leaving Ser-41 as the NH(2)-terminal residue of the processed TSA protein. These results provide strong evidence that the first 45 and 40 amino acid residues of PAI and TSA, respectively, are functional transit peptides (Fig. 2A).


Figure 7: Radioactive sequencing of mature PAI and TSA proteins processed by isolated chloroplasts. The PAI (A) and TSA (B) precursors were labeled with [S]methionine and [^3H]leucine and processed into their mature sizes by isolated spinach chloroplasts. The mature form proteins were subjected to automated Edman degradation, and the radioactivity of each cycle was counted. Thick and thin arrowheads indicate the expected positions of [S]methionine and [^3H]leucine, respectively. The predicted sequences of processed PAI and TSA are indicated in one-letter symbols with labeled methionine and leucine in bold letters.



Localization of Tryptophan Biosynthetic Enzymes in Isolated Arabidopsis Intact Chloroplast

Although the in vitro import studies are consistent with the presence of the Arabidopsis tryptophan pathway proteins in the chloroplast, this hypothesis was directly tested for the five proteins against which antisera were raised. Intact leaf chloroplasts of Arabidopsis were prepared by Percoll gradient centrifugation. To estimate the proportion of the tryptophan biosynthetic proteins present in the chloroplasts relative to whole leaf extract, equivalent amounts of chloroplast protein from leaf and intact chloroplast (as judged by their chlorophyll content) were subjected to immunoblot analysis. Within experimental error, all of the ASA, PAT, PAI, TSA, and TSB proteins present in the crude extracts were judged to be in the intact chloroplasts (Table 1). These results indicate that the majority of the tryptophan biosynthetic enzymes present in the whole leaf extract are found in Arabidopsis chloroplasts.




DISCUSSION

In this paper, we report the production and characterization of polyclonal antibodies against the tryptophan biosynthetic proteins PAT, PAI, TSA, and TSB from Arabidopsis. These antibodies were specific for the targeted proteins based upon several lines of evidence. First, each antibody recognized only one major protein band in the expected molecular weight range on an immunoblot of Arabidopsis leaf protein extract. Second, the antibodies recognize proteins in E. coli when the bacteria are transformed with the cognate Arabidopsis cDNA clone. Third and most significantly, Arabidopsis mutants defective in the structural genes for PAT, TSA, or TSB showed significant reduction in the amount of corresponding cross-reactive material. Although no Mendelian mutant is available for PAI, the antibody against PAI appears to be specific for its protein in Arabidopsis extracts because the cross-reacting material was greatly reduced in transgenic Arabidopsis plants that express a PAI antisense RNA construct.^4

Antibody against Arabidopsis ASA was also generated. Although neither Arabidopsis ASA-deficient mutants nor antisense plants are available, our data indicate that anti-ASA is specific for the ASA protein. The evidence for this assertion is 2-fold: there is a single major immunoreactive protein band detected in Arabidopsis leaf protein extracts, and the mobility of this protein is consistent with reports on the sizes of plant ASA proteins from Ruta graveolens (60 kDa), (^8)Catharanthus roseus (67 kDa; (21) ), and Zea mays (61.5 kDa; (18) ).

Higher molecular weight Arabidopsis PAI and TSA precursor proteins were imported into and processed by chloroplasts in vitro. The imported mature PAI and TSA proteins are likely localized in the stroma of chloroplast because they were protected from protease treatment and found to be highly enriched in the soluble fraction of the chloroplasts. The antisera allowed us to directly demonstrate that isolated intact Arabidopsis chloroplasts contained more than 90% of the immunologically detectable tryptophan pathway proteins of Arabidopsis leaves. These results strongly support the previously proposed chloroplast localization of the pathway(8, 9) . NH(2)-terminal sequencing data indicate that the cleavage sites of PAI and TSA precede the regions where the Arabidopsis sequences start to align with known microbial sequences. The calculated molecular mass of the processed TSA protein (28.8 kDa) is in good agreement with the apparent molecular mass based on SDS-PAGE (31 kDa). In contrast, the mobility of both the precursor (34 kDa) and mature (31 kDa) forms of PAI are slower than expected based upon the deduced amino acid sequence (29.6 and 24.7 kDa, respectively). This reproducible difference appears to be caused by an electrophoresis artifact.

Precursors of PAT, PAI, and TSA appear to be digested by E. coli proteases to smaller proteins including forms close in size to the mature plant proteins (Fig. 3). When expressed in E. coli, potato 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and Arabidopsis acetohydroxyacid synthase precursors, both chloroplast proteins, were also processed to their mature sizes(22, 23) . One possible explanation for the accumulation of the mature-sized proteins in E. coli is that the mature protein portion of the precursor folds into its native form leaving the transit peptide susceptible to protease digestion. We cannot rule out the possibility that E. coli contains a peptidase with a specificity similar to that of the chloroplast transit peptidase.

No significant amount of PAT precursor was found in E. coli expressing PAT cDNA. Instead, it appears that the E. coli proteases produce a variety of smaller fragments, including a fragment that co-migrates with mature PAT protein (Fig. 3A). Similar results were obtained by in vitro synthesis of PAT protein: although a higher molecular weight PAT precursor was obtained, the majority of the in vitro labeled proteins are smaller fragments of PAT protein (data not shown). The PAT precursor protein may be unstable when produced in E. coli or in the reticulocyte translation system. Premature termination is another possible explanation for the smaller fragments.

As tryptophan is used for protein synthesis in the cytosol and mitochondria as well as in the plastid, the localization of the tryptophan biosynthetic pathway in chloroplasts of plants implies an efficient transport system for tryptophan across the chloroplast membranes. In good agreement with this assumption, approximately 90% of the tryptophan synthesized by isolated chloroplasts incubated with [1,6-^14C]shikimic acid was found free in the reaction medium rather than within chloroplasts(24) . Because Arabidopsis anthranilate synthase, the committing enzyme of the pathway, is feedback-inhibited by tryptophan(25) , the localization of this protein within the chloroplasts suggests that the intraplastidic tryptophan concentration directly influences anthranilate synthase activity. Therefore, the concentration of tryptophan inside the chloroplasts might be regulated by the demand for tryptophan of the entire cell. The implications of the localization of the tryptophan biosynthetic enzymes in the chloroplasts on the regulation of the pathway deserve further study.


FOOTNOTES

*
This work was supported by Grant GM43134 from the National Institutes of Health and a National Science Foundation Presidential Young Investigator Award (to R. L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
E. R. Radwanski and R. L. Last, unpublished results.

(^2)
Barczak, A., Zhao, J., Pruitt, K. D., and Last, R. L.(1995) Genetics, in press.

(^3)
E. R. Radwanski, J. Zhao, and R. L. Last, submitted for publication.

(^4)
Li, J., Zhao, J., Rose, A. B., Schmidt, R., and Last, R. L.(1995) Plant Cell, in press.

(^5)
The abbreviations used are: ASA, anthranilate synthase alpha subunit (EC 4.1.3.27); PAT, phosphoribosylanthranilate transferase (EC 2.4.2.18); PAI, phosphoribosylanthranilate isomerase; TSA and TSB, tryptophan synthase alpha and beta subunits (EC 4.2.1.20); GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

(^6)
A. B. Rose and R. L. Last, manuscript in preparation.

(^7)
A. B. Rose and R. L. Last, unpublished results.

(^8)
J. Bohlmann and W. Martin, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Paul Bernasconi and Mani Subramanian for providing GST-ASA fusion protein, members of the laboratory for the plasmids used in this study, the Center for Research Animal Resources at Cornell University for antibody production, and the Cornell Biotechnology Analytical/Synthesis Facility for protein sequencing. We thank Klaus M. Herrmann, Dan J. Kliebenstein, and Elaine R. Radwanski for helpful comments on the manuscript.


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