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(Received for publication, December 8, 1994; and in revised form, December 23, 1994) From the
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
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
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 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
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.
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.
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. (
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.
Figure 5:
In vitro processing and uptake of
the Arabidopsis PAI and TSA precursors into isolated spinach
chloroplasts. [
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
[
Figure 7:
Radioactive sequencing of mature PAI and
TSA proteins processed by isolated chloroplasts. The PAI (A)
and TSA (B) precursors were labeled with
[
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. 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), ( 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 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-
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6081-6087
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit, phosphoribosylanthranilate
transferase, phosphoribosylanthranilate isomerase, and the tryptophan
synthase and 
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
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.
subunit(4) , trp1 mutants lack
phosphoribosylanthranilate transferase
activity(5, 6) , and trp3 and trp2 mutants have defects in the tryptophan synthase (
)and (7) (
)subunits, respectively.
termini(3, 4, 6, 7, 12, 13) . (
)(
)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
activity(7) . We are unaware of any other direct biochemical
demonstration that tryptophan biosynthetic enzymes are present in
chloroplasts. subunit (ASA), (
)phosphoribosylanthranilate transferase (PAT),
phosphoribosylanthranilate isomerase (PAI), as well as the tryptophan
synthase (TSA) and 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-terminal sequencing of the
processed proteins revealed the location of the cleavage sites.
Materials and General Procedures
Escherichia
coli strain DH5 [supE44 
lacU169
(
80 lacZ M15) 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,
[H]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) (
)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) and trp3-1 (mutation in TSA1 gene) 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, 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. 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
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 Tris
HCl, 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 TrisHCl, 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, 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 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 SDS sample buffer
directly to the various samples.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 TrisHCl,
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) 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
[H]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.
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 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 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-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) .
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 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 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.
)When
anti-TSB was used to probe the TSB mutant trp2-8, 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. 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.
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.
S]Methionine- and
[H]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.
S]methionine and
[H]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
To define the precise cleavage sites in
the PAI and TSA precursors, the NH Termini of the
Processed PAI and TSA termini of the in
vitro processed PAI and TSA were determined. Fig. 7shows
the radioactive sequencing profiles of processed PAI and TSA labeled
with [H]leucine and
[S]methionine. Cycle 21 of PAI is high in H 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 (H) 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-terminal residue of the processed PAI protein.
Cycles 2, 9, and 11 of TSA sequencing are high in H
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-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).
S]methionine and
[H]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
[H]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.
)Catharanthus roseus (67 kDa; (21) ), and Zea mays (61.5 kDa; (18) ).-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.
C]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.
))))) subunit (EC 4.1.3.27); PAT, phosphoribosylanthranilate
transferase (EC 2.4.2.18); PAI, phosphoribosylanthranilate isomerase;
TSA and TSB, tryptophan synthase and subunits (EC
4.2.1.20); GST, glutathione S-transferase; PAGE,
polyacrylamide gel electrophoresis.)))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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