(Received for publication, August 10, 1995)
From the
A detailed comparison of citrate uptake into the vacuole-like
lutoids of rubber tree (Hevea brasiliensis Muell. Arg.) and of
malate and citrate transport into barley (Hordeum vulgare L.)
vacuoles revealed very similar transport specificities. In order to
identify proteins mediating the transport, two photoreactive analogues (N`-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic
acid and 5-azidoisophthalic acid) of malate/citrate were synthesized
and found to efficiently inhibit citrate uptake into barley vacuoles (K = 18 µM) and Hevea lutoid vesicles (K
= 27 µM). In vacuoles from both plant
species, these photoaffinity probes specifically labeled a single
protein with a molecular mass of 23.6 kDa. This citrate binding protein
(CBP) was purified to homogeneity from Hevea lutoids, and
amino acid sequences were determined for NH
-terminal and
tryptic peptides. Using degenerate oligonucleotides of the
NH
-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of
238 amino acids, containing an NH
-terminal 31-amino acid
signal sequence for endoplasmic reticulum targeting, a prerequisite for
vacuolar localization. The mature CBP does not show significant
sequence similarities to any known primary protein structure and thus
represents a member of a novel class of proteins.
Malate and citrate play central roles in plant metabolism as
intermediates of the TCA cycle and of CO fixation, as
components of pH homoeostasis and as the transport forms of reducing
equivalents (for review, see (1) ). When accumulating at
elevated levels in plants, these acids have been shown to reside in the
vacuole. Oleski et al.(2) investigated the transport
of citrate into tonoplast vesicles from tomato fruits and observed that
citrate uptake was inhibited by malate and other dicarboxylic acids.
Lutoids, which are vacuole-like vesicles in the latex of the rubber
tree (Hevea brasiliensis), have been shown to accumulate
citrate in vivo(3) (5.7 mM and 53 mM citrate in cytoplasm and lutoids, respectively). Citrate transport
into lutoids in vitro was first described by d'Auzac and
Lioret (4) using intact native lutoids, and later by Marin (5) using vesicles reconstituted from freeze-dried lutoids.
Both native lutoids and tonoplast vesicles were shown to accumulate
citrate from the outside against a concentration gradient. The uptake
was stimulated by MgATP, and inhibitors of the lutoid tonoplast ATPase
inhibited this activation of citrate
uptake(5, 6, 7) . Interestingly, the two
dicarboxylates, malate and succinate, appear to cross the membrane by
the same, or a closely related, transporter(8) . In
photosynthesizing protoplasts from barley leaves, malate and citrate
are rapidly transferred to the vacuole(9) . Transport of malate
across the vacuolar membrane is not specific for the natural
enantiomer, L-malate, since D-malate, citrate, and
other di- and tricarboxylic acids behaved as competitive
inhibitors(10, 11, 12) . Specific inhibition
of the ATPase indicated that malate transport occurs by a secondary
active transport mechanism(13) . A detailed comparison of the
uptake of citrate and malate into barley vacuoles showed that the
characteristics of uptake are very similar and this suggested that
malate and citrate are transported by the same transport system across
barley tonoplasts(14) . However, in order to determine the
substrate specificity in detail and, furthermore, to understand the
regulation of malate and citrate transport into vacuoles, the
identification of the protein(s) mediating malate and citrate uptake is
required. The malate/citrate carrier of barley tonoplasts (15) and the malate transporter of Kalanchoë daigremontiana vacuoles (16) have previously been functionally reconstituted and
partially purified. The reconstituted carriers showed properties
similar to those described for the carrier of intact vacuoles. However,
further attempts to purify and identify the proteins mediating this
transport activity were unsuccessful to date, due to limitations in the
availability of tonoplast material and to the lack of methods for their
detection.
In this study, we have compared citrate uptake into barley vacuoles and Hevea lutoid vesicles and were able to show that transport is efficiently inhibited by two photoreactive dicarboxylates. These photoaffinity probes specifically label a 23.6-kDa protein in both plant systems. We describe the isolation of the citrate binding protein of Hevea, the isolation of the corresponding cDNA and its gene, as well as the identification of homologous genes in barley.
Aliquots of frozen Hevea lutoid fractions were thawed on
ice in medium A (300 mM mannitol, 25 mM Mes, ()25 mM Mops, 25 mM Hepes, adjusted to pH
6.0 with Tris base(6) ). The suspension was homogenized on ice
using a glass homogenizer (1 g bottom fraction per 10 ml of medium) and
vesicles were sedimented by centrifugation at 10,000
g for 15 min at 4 °C. The pellet was resuspended and centrifuged
twice under the same conditions. To avoid contamination of lutoid
vesicles by soluble vacuolar compounds, additional homogenization and
washing steps were performed, but these affected neither the protein
composition visualized on silver-stained gels nor the labeling pattern.
Finally, the sediment was resuspended in an equal volume of medium B
(the same as medium A, but adjusted to pH 7.5 with Tris base) and used
for tracer uptake experiments, for further purification, or for
photoaffinity labeling experiments.
To energize the accumulation of citrate in proteoliposomes, a
H gradient was generated by alkalinization of the
medium. For this, one part of medium D (300 mM mannitol, 75
mM Tricine, pH 8.5, plus 10 µl of 10 N KOH per 10
ml of medium) was added to two parts of proteoliposomes resulting in a
proton gradient of approximately
pH 2.8.
The assay medium for
citrate uptake contained 2.2 kBq of [C]citrate
per 100 µl of assay medium (43.3 MBq/mmol) and solutes as
indicated. After adjusting the concentration of organic acids to 10
mM (with citrate), proteoliposomes were separated from the
incubation medium by a Dowex 1X8-100 column(15) .
Hevea lutoid vesicles were prepared as
described for the uptake experiments. Concentration of the
photoaffinity label was 15-20 µM. Photolysis of the
azido derivatives was achieved by illuminating the sample in microtiter
plates, placed on ice, with UV light of max 366 nm (Camag; 420
milliwatts/cm
). Illumination time was 5 min.
Figure 6:
Complete cDNA sequence and deduced amino
acid sequence of HbCBP. Fragments were assembled as shown in Fig. 5. The NH-terminal amino acid sequence and
tryptic peptides sequences determined by amino acid sequence analysis
of the mature protein are underlined. Putative polyadenylation
signals are typed bold.
Figure 5:
Schematic overview of cDNA and genomic
clones encoding rubber tree CBP. NH-terminal (227 bp) and
COOH-terminal (630 bp) cDNA fragments are shown in the upper
part, the genomic 1.4-kb PCR fragment, which links the two cDNA
fragments at the EcoRI site, is shown at the bottom.
A fragment of the HbCBP gene was amplified using inverse PCR. Genomic DNA from young Hevea leaves (20) was digested with PstI, circularized with T4 DNA-ligase, and used as template for the primers Hb3 5`-CAGAGCAAAAATGTCTGTCC corresponding to position 119 to 100 (cDNA sequence in Fig. 6) and Hb4 5`-GATCCAACTGATGGGTTCAC corresponding to positions 122 to 141. An aliquot was used as template for a second PCR using primers Hb5 5`-GCTCACAAGAAGCAAAAGGG corresponding to position 91 to 72 and Hb6 5`-TGAGGTGCCATTAACAGAGG corresponding to positions 142 to 161. The single amplified fragment of about 1.4 kb was isolated, subcloned, and sequenced.
Figure 1: Structure of HABDA (A) and AIPA (B). The position of the tritium label is marked by an asterisk.
Barley tonoplasts were isolated and the proteins subjected to
chromatography on dry hydroxylapatite, after which malate and citrate
transport can still be measured in a reconstituted system(15) .
This purified protein fraction was irradiated in the the presence of
[H]AIPA and in the presence or absence of
competing citrate. A specifically labeled protein with an apparent
molecular mass of 27 kDa was identified after SDS-PAGE and fluorography (Fig. 2). Since binding of the photoaffinity probe could be
prevented by addition of 10 mM citrate before irradiation, the
labeled protein was a good candidate for a putative malate and citrate
transporter. However, for purification of the labeled polypeptide and
for more detailed analyses of binding of the photoaffinity probes, the
availability of barley tonoplasts was limiting. Vacuoles from Hevea latex seemed to be an advantageous source of tonoplast material,
since large amounts can be isolated with a minimum of preparative
efforts. To demonstrate similarity to the described transport systems
in barley mesophyll vacuoles, it was, however, necessary to
characterize citrate transport specificity into lutoid vesicles in more
detail.
Figure 2:
Photoaffinity labeling of barley tonoplast
proteins partially purified by chromatography on hydroxylapatite with
[H]AIPA. Purified barley tonoplasts
(corresponding to 1.55
10
vacuoles) were irradiated
for 5 min at 366 nm in the absence(-) or presence (+) of 10
mM citrate and proteins subsequently separated by SDS-PAGE. In A the gel stained with silver nitrate is shown; in B the corresponding fluorogram is presented. The fluorogram was
exposed for 6 months. The molecular masses of marker proteins are given
in kDa.
Figure 3:
Photoaffinity labeling of Hevea lutoid vesicles with [H]HABDA. Lutoid
vesicles corresponding to 35 µg of protein were irradiated for 5
min at 366 nm in the absence(-) or presence (+) of 10 mM citrate and subsequently separated by SDS-PAGE. In A the
gel stained with silver nitrate is shown; in B the
corresponding fluorogram is presented. The fluorogram was exposed for
10 days. The molecular masses of marker proteins are given in
kDa.
To find out whether the labeled polypeptide was an integral
component of the lutoid membrane, loosely associated membrane proteins
were removed by repeatedly freezing and washing the vesicles with 0.5 M KCl or by washing with 0.2 M
NaCO
(21) . After KCl washes most of the
labeled protein was retained in the membrane fraction. However, washes
with Na
CO
released part of the CBP into the
supernatant. But even after Na
CO
washing
labeled CBP can still be detected in the membrane fraction (not shown),
indicating that although the labeled protein is tightly associated with
the lutoid membrane, the CBP is probably not an integral membrane
protein.
To determine the specificity of binding of the
photoaffinity probe, di- and tricarboxylates and other anions were
tested for their ability to prevent binding of
[H]HABDA to CBP (Table 1, B). Proteins were
separated by SDS-PAGE, gels were subjected to fluorography, and the
intensity of labeling was determined by densitometric measurements.
Citrate, isocitrate, isophthalate, HABDA, and benzenetricarboxylate
inhibited binding of the photoaffinity label very efficiently, whereas
malate and succinate were less effective. Oxaloacetate,
-hydroxybutyrate, chloride, sulfate, and nitrate (not shown) at
the same concentration did not have any significant effect. With the
exception of isocitrate, which prevents efficient binding of the
photoaffinty label but is only slightly inhibitory in the uptake
experiments, inhibition of uptake agreed very well with protection from
affinity labeling. To determine whether the labeled protein was able to
mediate citrate transport, the CBP of Hevea was purified and a
reconstitution system was established which allowed measurement of
citrate transport activity.
Figure 4:
Purification of CBP of Hevea and
labeling of the purified CBP with [H]HABDA.
Lutoid vesicles (1) and the purified protein (2)
separated by SDS-PAGE and stained with silver nitrate. Fluorogram of
purified CBP labeled with [
H]HABDA prior gel
electrophoresis in the absence (3) or presence (4) of
10 mM citrate. Molecular masses of marker proteins are given
in kDa.
Figure 7:
Analysis of leader peptide and hydropathy
plot of the deduced amino acid sequence of CBP of Hevea.A, the NH-terminal sequence as determined by
amino acid analysis of the mature protein is shown by italics in the bottom line. The top line shows the
deduced NH
-terminal sequence starting from the first ATG in
the open reading frame. Positive charges and the hydrophobic core are
marked. B, hydropathy plot calculated from the complete amino
acid sequence of HbCBP. The analysis was performed according
to Kyte and Doolittle (24) with a window of 11 amino
acids.
A data base search for
related protein sequences revealed no significant similarities to known
primary protein structures. Analysis of the hydrophobicity of the
deduced amino acid sequence demonstrates that CBP is hydrophilic and
contains only one hydrophobic stretch which might not be sufficient to
act as a membrane spanning domain (Fig. 7B)(24) . Predictions for the secondary
structure indicate that no amphipathic helices large enough to span the
lipid bilayer are likely to be present in CBP. The hydrophobic
NH terminus of the primary translation product cannot be
used for integration into the membrane since the signal peptide is not
present in the mature protein.
Figure 8: Southern blot analysis of genomic DNA of Hevea (A) and barley (B). High molecular weight DNA was digested with the restriction enzymes HincII, BglII, BamHI, HindIII, EcoRI, XbaI, PstI, and NsiI and subjected to Southern blot analysis using labeled 630-bp COOH-terminal cDNA fragment as probe. 25 µg of DNA were loaded per lane. Size markers are given in kilobases.
The concentration of malate increases in C plants
during light-dependent nitrate reduction, and in plants exhibiting CAM
metabolism malic acid accumulates as a store for CO
during
the night. However, the concentration of malate in the cytosol remains
rather constant and excess malate is transferred rapidly into the
vacuole, where it accumulates to considerable levels (for review, see (1) ). Diurnal fluctuations of malate in C
and CAM
plants thus predominantly represent changes in vacuolar malate
concentration and require malate transport across the tonoplast.
Besides malate, citrate levels are subject to substantial day-night
changes in CAM plants(25) . Vacuolar malate and citrate
transport have been shown in a variety of species and have been
characterized at the biochemical level in barley vacuoles and tonoplast
vesicles of CAM plants(10, 12) . In barley vacuoles,
citrate and malate appear to be transported by the same
system(14) . Purification of the proteins mediating the
transport of malate and citrate has been limited due to the
difficulties in obtaining tonoplasts from barley vacuoles in sufficient
quantities. Hevea lutoids represent an attractive source for
the purification of tonoplast proteins since they can efficiently be
isolated from latex sap. Previous investigations on citrate uptake into
lutoids focused mainly on the energetics of transport(26) . The
detailed comparison presented here has now revealed that citrate
transport into Hevea lutoid vesicles exhibits characteristics
similar to those of malate and citrate uptake into barley vacuoles.
Analysis of its hydrophobicity indicates that CBP does not represent an integral membrane protein with multiple hydrophobic domains. This is in agreement with biochemical data showing that CBP is loosely associated with the lutoid membrane. In a parallel case, an affinity probe for sucrose allowed the identification of a hydrophilic 62-kDa sucrose binding protein (35) which is associated with the plasma membrane and appears to be involved in sucrose transport. The low hydrophobicity of CBP indicates that it does not represent the actual pore-forming subunit of the putative citrate transporter. However, binding of the photoaffinity probe and inhibition characteristics of citrate transport suggest that CBP may represent a peripheral subunit involved in substrate recognition. Of the few tonoplast proteins identified to date, the vacuolar ATPase is an example of a heteromultimeric complex (36) . In Escherichia coli, amtA probably represents a cytoplasmic component of the ammonium transporter(37) . Periplasmic proteins responsible for binding and subsequent transport of substrates (38) are required for the uptake of various solutes and provide further examples for transport not being mediated by a single polypeptide.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89855[GenBank].