(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 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.
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
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.
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 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.
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 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.
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. ()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.
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.
Figure 5:
In vitro processing and uptake of
the Arabidopsis PAI and TSA precursors into isolated spinach
chloroplasts. [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.
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
[
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.
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
[
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.
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), ()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-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-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.