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J Biol Chem, Vol. 273, Issue 46, 30568-30575, November 13, 1998
From the Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
A cDNA for a water-soluble chlorophyll (Chl)
protein (WSCP) from cauliflower (Brassica oleracea L. var
botrys) was cloned and sequenced. The cDNA contained an
open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP. The sequence showed extensive
homology to drought-stress-related, 22-kDa proteins in some
Brassicaceae plants. Functional WSCP was expressed in Escherichia
coli as a fusion protein with a maltose-binding protein (MBP).
When the recombinant MBP-WSCP was incubated with thylakoid membranes,
the MBP-WSCP removed Chls from these membranes. During this process,
the monomer of the apo-MBP-WSCP successfully bound Chls and was
converted into tetrameric holo-MBP-WSCP. The reconstituted MBP-WSCP
exhibited absorption and fluorescent spectra identical to those of the
native WSCP purified from cauliflower leaves. The Chl
a/b ratio in native WSCP indicates a high
content of Chl a, which was mainly due to the higher
affinity of MBP-WSCP for Chl a. WSCP is the first example
of a hydrophilic protein that can transfer Chls from thylakoid
hydrophobic proteins. Possible functions of WSCP are discussed.
A water-soluble chlorophyll protein
(WSCP)1 in Brassicaceae
plants has been purified from Lepidium virginicum (1),
Brassica nigra (wild mustard) (1), Brassica
oleracea var. botrys (cauliflower) (2, 3),
Brassica oleracea var. gemmifera (Brussels
sprouts) (4), and Brassica napus var. oleifera
(rapeseed) (3). Although all these WSCPs exhibit similar properties in
terms of hydrophilicity, molecular size, and subunit conformation, they
can be categorized into two groups on the basis of their chlorophyll
content. The first group shows a higher content of Chl a,
and includes WSCPs from cauliflower (Chl a/b = 6.3) (2), wild mustard (Chl a/b = 8) (1),
and Brussels sprouts (Chl a/b > 10) (4);
the second group is characterized by a higher content of Chl
b, as in WSCPs from L. virginicum (Chl
a/b = 1-2) (5, 6).
Although some physicochemical and biochemical characterizations of
WSCPs have been performed, almost nothing has been determined in regard
to the physiological function of these proteins in plant tissues. In
recent studies, it was suggested that the WSCPs from cauliflower (3)
and Brussels sprouts (4) may be identical to a drought-induced 22-kDa
protein in rapeseed, BnD22, which possesses a motif of the
Künitz-type proteinase inhibitor family (7, 8). Although WSCPs
from cauliflower (3), rapeseed (3), Brussels sprouts (4), and L. virginicum (Y. Oka, K. Fujiyama, H. Satoh, K. Nakayama, and M. Okada (1996) DDBJ accession no. AB002589) possess
this motif, those from cauliflower and Brussels sprouts have been
reported not to inhibit trypsin (3, 4). On the other hand, it has been
speculated that BnD22 controls proteolytic pathways in plants, based on
its ability to slightly inhibit chymotrypsin activity (10).
In addition to their predicted function as a proteinase inhibitor,
WSCPs have been postulated to function as a Chl carrier (4, 5).
However, there are no experimental data in support of this hypothesis.
In this study, we have cloned the cauliflower WSCP cDNA and
expressed it in Escherichia coli as a fusion protein linked
to maltose-binding protein (MBP). The recombinant MBP-WSCP successfully
bound Chl molecules in the same fashion as did the native WSCP when the
protein was mixed in aqueous solution with thylakoid membranes from
spinach leaves. This result makes WSCP the first documented hydrophilic
protein capable of removing Chls from thylakoid pigment proteins, and
suggests that the WSCP may function as a Chl carrier in plants.
Plant Material and Purification of Native WSCP--
Seeds of
cauliflower (B. oleracea L. var. botrys) were
germinated on cubes of glass fiber irrigated with half-strength
Hoagland's solution. Seedlings were grown hydroponically with the same
medium in a greenhouse under a 16-h photoperiod at 23 °C. The volume of medium was maintained at the original level by addition of water
every other day, and the culture medium was changed every 2 weeks.
Cauliflower plants for purification of native WSCP were obtained from a
local farmer in Chiba, Japan. About 2 kg of cauliflower leaves were
used for the WSCP purification (3). The molecular weight of native WSCP
was determined by mass spectrometry (Voyager; PerSeptive Biosystems).
The protein concentration was determined by the bicinchoninic acid
method using a kit from Sigma. Bovine serum albumin was used as a standard.
Amplification and Cloning of the WSCP cDNA from Cauliflower
Leaves--
cDNA of cauliflower-WSCP was amplified by rapid
amplification of cDNA ends (RACE) (11). Two-month-old cauliflowers
grown hydroponically were transferred, along with their bases, from the
growth medium and left to dry for 2 days in the greenhouse. Total leaf
RNA was extracted by the guanidium thiocyanate method (12). First
strand cDNA was synthesized by reverse transcription of total RNA
by priming the reaction with an oligo(dT)20-M4 anchor (5'-GTTTTCCCAGTCACGACTTTTTTTTTTTTTTTTTTTT-3'). The
reverse-transcription reaction was performed at 42 °C for 1 h
with 3 µg of RNA and 300 units of reverse transcriptase XL (Takara
Biomedicals, Japan) in a 10-µl mixture.
Previously, 46 amino acid residues from the NH2 terminus of
native WSCP from cauliflower were determined (3). The amplification was
performed using Ex Taq DNA polymerase (Takara Biomedicals) in the presence of sense primer (5'-AGAGAACAGGTGAAGGA-3') corresponding to NH2-terminal residues REQVKD, and anchor
(5'-GTTTTCCCAGTCACGAC-3') primer. PCR (GeneAmp 2400; Perkin-Elmer) was
conducted for 30 cycles using the profile: 94 °C for 2 min, 50 °C
for 2 min, and 72 °C for 2 min. An additional 10 min at 72 °C was
used for the last cycle.
The PCR product was cloned into pBluescript II KS(+) vector
(Stratagene) at the EcoRV site. Six independent recombinant
clones were sequenced using an ALFred DNA sequencer with a Thermo
Sequenase or an Autoread DNA Sequencing Kit (all from Amersham
Pharmacia Biotech).
The nucleotide sequence of the cDNA confirmed that it encoded WSCP
of cauliflower and BnD22 of rapeseed (3, 7, 8). For further
confirmation of the identity of the cDNA, we performed RACE-reverse
transcription PCR with a sense DNA primer (5'-ATGAAGACTTTTTTTCTAG-3') corresponding to the NH2-terminal residues of the BnD22
precursor, MKTFFL (7). An amplified fragment was cloned and sequenced.
Expression of Recombinant MBP-WSCP in E. coli--
To express
the mature form of WSCP, a cDNA without the signal peptide was
excised and cloned into a procaryotic expression vector, pMAL-cRI (New
England Biolabs). Cauliflower WSCP was expressed in E. coli
as a fusion protein linked to the MBP. Expression of MBP-WSCP and
affinity purification by amylose column chromatography were performed
according to the manufacturer's instructions. Profiles of the
expressed protein and its purification were monitored by SDS-polyacrylamide gel electrophoresis (PAGE) (13).
Reconstitution of the MBP-WSCP with Chls by Mixing with Thylakoid
Membrane Preparations--
Thylakoid membranes were prepared from
spinach chloroplasts according to the methods of Nakayama et
al. (14). Five nmol of WSCP was mixed with membrane aliquots
containing 0, 1.25, 5 or 20 nmol of Chls in an aqueous solution of 10 mM sodium phosphate buffer (pH 7.2), 0.5 M
NaCl, 1 mM EDTA, and 10 mM maltose. After 30 min at 22 °C, the water-soluble fraction was recovered by
centrifugation at 10,000 × g for 30 min and subjected
to PAGE (detergent-free) using an 8% polyacrylamide gel. Fluorescence
of Chls bound to MBP-WSCP was detected on a long wave UV
transilluminator. Protein bands were visualized by Coomassie Brilliant
Blue (CBB) staining.
To analyze the spectrophotometric properties of the reconstituted
MBP-WSCP, thylakoid membranes with an 8-fold molar excess of Chls
relative to MBP-WSCP were mixed with MBP-WSCP. After centrifugation, the water-soluble fraction, as well as native WSCP purified from cauliflower, were analyzed by absorption and fluorescence
spectrometries using a U-3300 spectrophotometer (Hitachi Co., Ltd.) and
a FluoroMax spectrofluorometer (SPEX Industries). The absorption
spectral data at 0.2-nm intervals were analyzed as fourth derivative
spectra (15). Spectra were resolved into their components (16) using an
Igor Multi-peak Fit package (Wave Metrics, Inc.).
To determine the ratio of Chl molecules to MBP-WSCP, a thylakoid
membrane preparation containing 200 nmol of Chls was incubated with 40 nmol of MBP-WSCP at 23 °C for 30 min and centrifuged (10,000 × g, 15 min). An aliquot of the supernatant was then subjected to protein quantitation. SDS solution was added to the remaining supernatant to a final concentration of 1%. Chls were extracted from
holo-MBP-WSCP by diethyl ether, and the concentrations of Chls were
determined spectrophotometrically in 80% acetone (17). The
stoichiometry was calculated from the amounts of protein and Chls in
reconstituted MBP-WSCP.
Reconstitution of the MBP-WSCP with Purified
Chls--
Photosynthetic pigments were extracted from spinach leaves
with chloroform (18) after the leaves were dried by grinding with a
silica gel (19). Chls a and b were separated by
reversed-phase high performance liquid chromatography (Unicil Pack C18;
Gasukuro Kogyo, Japan) using 100% methanol as an eluent.
Five hundred pmol of MBP-WSCP was mixed with 0, 12.5, 125, 250, or 500 pmol of purified Chl a in 20% ethanol and 0.1 M
sodium phosphate buffer (pH 7.2). The mixtures were incubated at
22 °C for 30 min and subjected to 8% PAGE (detergent-free). The
Chls and proteins were visualized.
Molecular Cloning of a cDNA for Cauliflower WSCP and Analysis
of Its Deduced Amino Acid Sequence--
A cDNA for cauliflower
WSCP was amplified by 3'-RACE PCR with a sense primer deduced from
residues 1-6 (REQVKD) of the native WSCP (3), and cloned. Six
independently isolated recombinants contained identical sequences.
These 729-base pair cDNAs contained an open reading frame encoding
199 amino acids, and a 3'-untranslated region of 129 bases. All 48 amino acids of the native WSCP (3) corresponded to the deduced amino
acid sequence of the cDNA starting at residue 1 of the mature form
of WSCP.
Our previous work suggested that cauliflower WSCP and BnD22, a
drought-induced protein in rapeseed, were identical (3). To verify this
identity, we used a sense primer corresponding to the NH2
terminus of a precursor of BnD22 for the RACE-PCR with cauliflower RNA.
An amplified band was cloned and sequenced (Fig. 1). The sequence of the cDNA was
identical to that of the fragment amplified with a sense primer for the
NH2 terminus of mature WSCP. In addition, we cloned a
cDNA for BnD22 from rapeseed and expressed it in E. coli. The recombinant MBP-BnD22 exhibited the same properties of
binding to Chl as did the recombinant MBP-WSCP (discussed below). Together, these results indicate that the drought-induced BnD22 protein
in rapeseed is WSCP.
Sequence Comparisons of WSCP to Its Homologs in Brassicaceae
Plants--
The deduced amino acid sequence of the cDNA was
compared with a data base by the BLASTp program (20). Sequences of
proteins in Brassicaceae with high homology to WSCP are aligned in Fig. 2. All proteins listed in the figure
contain a motif for the Künitz-type proteinase inhibitor family
(PROSITE (21) accession no. PS00283, indicated by the thick
bar in Fig. 2), although the functional significance remains
obscure.
The WSCP sequence of cauliflower (B. oleracea) shows
extensive homology (94%) to BnD22 of rapeseed (B. napus),
supporting the identity of both proteins. In contrast, the WSCP in
L. virginicum (DDBJ accession no. AB002589)
showed a much lower homology (44%) to that from cauliflower. In
Raphanus sativus, a cDNA encoding a protein that is 80%
homologous to WSCP, designated P22, has been cloned as a salt- and
drought-stress-inducible protein (22). Similar drought-related proteins
have been cloned from Brassica rapa (C. Shin, S. Song, and
Y. Choi (1993) GenBank accession no. Z25770) and Arabidopsis
thaliana (24). The sequence homologies of cauliflower WSCP to
these two proteins are 51% and 26%, respectively. Although it is
unclear whether or not these proteins can bind Chls, it can be
concluded that proteins homologous to WSCP are widespread in Brassicaceae.
Residues that were conserved among the six proteins listed in Fig. 2
are indicated by asterisks. Many of these conserved residues were located in the NH2-terminal half, suggesting that the
functionally and/or structurally important regions might be located in
this region. A conserved region in WSCP,
39PFCPLGI45 (indicated by the thin
bar in Fig. 2), showed homology to the sequence motif called
the (F/Y)DPLGL motif (25) of light-harvesting Chl
a/b-binding protein and its related proteins,
e.g. 211PFDPLGL217 in CP29 of barley
(26). In a three-dimensional structural model of pea light-harvesting
Chl a/b-protein complex II (25), this motif is in
a region located at the top of the third helix and covers Chl
a1 and one head group of lutein (25). This suggests the
possibility that this motif in WSCP might play a role in Chl binding.
Although the sequence homology in the COOH-terminal half is relatively
low, the COOH-terminal 11 amino acids, EKLGL(K/R)MFPFY are highly
conserved. Interestingly, we found that 10 COOH-terminal residues in
this hydrophobic region were missing in the mature form of WSCP. The
molecular weight of native WSCP, as determined by mass spectrometry,
was 20,226.4, which corresponds to the calculated molecular weight of
residues 1-189 (with an error of 0.048%) of the predicted 199-amino
acid sequence of mature WSCP. This conserved hydrophobic region may
function in targeting to a particular compartment; however, this region
may not affect the Chl-binding properties of WSCP, since the
recombinant MBP-WSCP that possesses this region still binds Chls
correctly (Figs. 4-9, discussed below).
Expression and Purification of Recombinant MBP-WSCP Fusion Protein
in E. coli Cells--
Recombinant WSCP was expressed in E. coli as a fusion protein with MBP. Fig.
3 shows the SDS-PAGE profiles of the
MBP-WSCP fusion protein during the expression and purification steps.
Addition of isopropyl-1-thio- Binding Activity of MBP-WSCP to Chls in Thylakoid
Membranes--
To examine the function of WSCP as a carrier of Chl,
MBP-WSCP was mixed in various ratios with thylakoid membranes (Fig.
4). When thylakoid membranes were not
added, a ladder of oligomeric MBP-WSCP formed (panel
A, lane 1), with the major form being
the monomer. When the MBP-WSCP was mixed with thylakoid membranes, the
major form became the tetramer, and the hexamer form was also apparent
(lanes 2-4 in panel A).
Interestingly, only the tetramers and hexamers emitted fluorescence
(panel B). These results indicate that the MBP-WSCP monomer
removed Chls from thylakoid membranes to form tetrameric and hexameric
structures. The dimeric form of apo-MBP-WSCP was also prominent
(lane 1 in panel A), but its level was not
substantially altered upon addition of thylakoid membranes. Further,
the dimer and trimer, unlike the tetramer and hexamer, did not emit
fluorescence. This raises the possibility that dimers and trimers may
be formed artificially (discussed below). Densitometric analysis of the
gel presented in panel A revealed that, prior to the
addition of Chl (lane 1), the ratio of
monomer:dimer was about 3:1. Forms other than monomers and dimers made
up less than about 3% of the total. Hence, at least 75% of the
MBP-WSCP in the preparation could bind Chl. This activity coefficient
of 0.75 was used in subsequent analyses of the data.
The stoichiometry of Chl in MBP-WSCP, which may be fully saturated with
Chl, was examined. A thylakoid membranes and MBP-WSCP were mixed with a
molar ratio of Chl:MBP-WSCP of 200:30. From the amount of protein and
Chl, the stoichiometry was calculated to be 2 Chls per tetramer, as in
the case of the native WSCP.
To compare the Chl-binding properties of the MBP-WSCP with those of the
native WSCP, absorption (Figs. 5 and
6) and fluorescence (Fig.
7) spectra were measured. The absorption
spectra of MBP-WSCP and native WSCP exhibited a similar shape in both
the red and Soret bands (Fig. 5A). MBP-WSCP showed a
shoulder at 485 nm, which was not as intense in the spectrum of the
native WSCP. This shoulder may represent one of the protein-bound forms
of Chl b, although this speculation remains to be verified.
The Chl a/b ratio in native WSCP displays
variations among the different WSCP preparations. Our previously
determined absorption spectrum for cauliflower WSCP possessed more Chl
b than the present spectrum (compare Fig. 5A with
Ref. 3). Diversity in the Chl a/b ratio has also
been reported in Lepidium WSCP (2, 5, 6).
Peak wavelengths of absorption spectra of each MBP-WSCP and native WSCP
in the red band were 672 and 673 nm, respectively (Fig. 5B).
The fourth derivative spectra (Fig. 5B) and a curve-fitting analysis of the red band of native WSCP (Fig. 6) deconvoluted it into
four major Chl a-components (Ca663, Ca671, Ca677, and Ca685)
and a single component for Chl b (Cb653) (Fig. 6). This deconvolution profile was almost identical to that for the native WSCP
purified from wild mustard (27). The intensity of each component in
both MBP-WSCP and native WSCP exhibited good agreement between the two
proteins with the exception of one component, Ca677 (Fig. 6). The
intensity of Ca677 was greater in native WSCP than in MBP-WSCP. This
difference caused a slight shift of the absorption peak between the two
spectra (Fig. 5B). However, in both proteins, peak
wavelengths of all Chl components were identical (Fig. 6).
Fluorescence emission spectra of both proteins excited at 440 nm (Fig.
7, panels A and B) and at 460 nm (Fig. 7,
panels C and D) were measured. These fluorescence
spectra were resolved into four major components assigned for Chl
a with peaks at 667, 672, 679, and 686 nm. Identical
emission peaks were obtained for native WSCP and MBP-WSCP at both
excitation wavelengths (Fig. 7, panels A and B,
C and D). When Chl a was excited, the
major fluorescence was emitted from Ca671 (F672) (panel A),
whereas excitation of Chl b gave a more intense signal from
Ca677 (F679) (panel C). Because very low level fluorescence
was emitted from Chl b, excited Chl b molecules
might efficiently transfer their energy to Chl a molecules
in the complex, as reported in WSCP from L. virginicum (28).
It should also be noted that considerable fluorescence was emitted from
all Chl a forms when Chl b was primarily excited,
suggesting that energy transfer also occurred between Chl a
molecules. These spectrofluorometric properties of the native WSCP were
exactly reproduced in the reconstituted MBP-WSCP (panels B
and D). However, it is possible that interaction of MBP-WSCP with thylakoid pigment proteins may not be perfectly comparable to that
of the native WSCP, because MBP-WSCP is much larger than the native WSCP.
Binding Activity of MBP-WSCP to Purified Chls--
The original
ratio of Chls a/b in thylakoid membranes from
spinach was 2.89. After Chls were extracted from the membranes by
mixing them with MBP-WSCP in a 0.75:4 molar ratio, the Chl a/b ratio in the remaining thylakoid membranes
decreased to 2.41. This indicates that MBP-WSCP preferentially removes
Chl a. In addition, native WSCP contains more Chl
a than Chl b (Fig. 4A). To address
whether this preference of Chl a is due mainly to the nature
of WSCP or to the properties of thylakoid membranes, the Chl-binding
activity of the MBP-WSCP was assayed using purified Chls a
and b (Fig. 8). Increasing
amounts of Chl a (lanes 1-5) or Chl
b (lanes 6-10) were mixed with MBP-WSCP. After
incubation at 22 °C for 30 min, proteins were separated on PAGE.
Proteins (panel A) and Chls (panel B) were
visualized as described above. In this experiment, the batch of
MBP-WSCP was different from that used in Fig. 4 and the dimeric form of
apo-MBP-WSCP (lane 1 in panel A) was not as
prominent; this supports the conjecture that the presence of the dimer
observed in Fig. 4A may be artifactual. Because the amounts
of forms other than the monomer of apo-MBP-WSCP were negligible, the
activity coefficient of this MBP-WSCP preparation was taken as 1.0.
Again, as shown in Fig. 8, the binding of purified Chls to MBP-WSCP
caused tetramer formation (panel A), and only the tetramer exhibited an intense Chl fluorescence (panel B). The binding
of Chl a to MBP-WSCP in a 0.5:1 molar ratio (lane
4 in panel A) resulted in almost no residual monomeric
form of the protein. The stoichiometry of Chl to the MBP-WSCP tetramer
was determined to be 2:1 (described above), suggesting that two
molecules of Chl a are sufficient to produce one MBP-WSCP
tetramer. In contrast, a 1:1 molar ratio of Chl b to
MBP-WSCP (lane 10 in panel A) did not eliminate
all of the monomeric MBP-WSCP, indicating that the MBP-WSCP possesses a
higher affinity for Chl a than for Chl b.
When MBP-WSCP was reconstituted with thylakoid membranes, the resulting
hexamer emitted more intense Chl fluorescence (Fig. 4B,
lane 4) than the hexamer formed during reconstitution with purified Chl (Fig. 8B, lane 5). Since different
batches of MBP-WSCP were used in these experiments, the same batch of
MBP-WSCP was used for the binding analysis to address the cause of this
difference (Fig. 9). Panel A
indicates CBB staining and panel B, visualization by
fluorescence. For an unknown reason, some of the oligomeric structures
showed split bands with CBB staining (panel A). However, reconstitution with thylakoid membranes led to the appearance of a
prominent hexameric band (lane 3 in panels A and
B), while less of the hexamer was formed when MBP-WSCP was
reconstituted with purified Chl a (lane 2 in
panels A and B). Of note was that reconstitution
with purified Chl a also yielded pentamers, heptamers, etc.,
which emitted fluorescence (lane 2 in panel B).
In contrast, no oligomeric forms other than the tetramer and hexamer
were detected during the electrophoretic purification of native WSCP
from cauliflower (data not shown). These results indicate that the
degree of hexamer formation is dependent on the substrate environment.
In thylakoid membranes, almost all Chls are bound with proteins (29),
whereas in ethanol solution, Chls are freely solubilized. Furthermore, other proteins in thylakoid membranes may affect the transfer. These
results suggest that reconstitution of MBP-WSCP with thylakoid membranes produces a WSCP quaternary structure identical to that of
native WSCP from cauliflower.
Intracellular Distribution and Possible Role of WSCP as a Putative
Chl Carrier--
The precursor of WSCP possesses 19 residues in the
NH2-terminal extension. Unexpectedly, this extension
possesses the signature of a signal peptide entering into a secretory
pathway (for review, see Ref. 30). In addition, the cleavage site of
the WSCP extension follows the "
On the other hand, it has been reported that native WSCP was detectable
in the water-soluble fraction of Percoll-purified class 1 chloroplasts
of L. virginicum (34). As described, this seems to conflict
with the notion that the NH2-terminal extension of
cauliflower WSCP may be a signal peptide. However, in some organisms,
several proteins are routed through the ER to plastids (35-37).
Therefore, it is possible that WSCP is transported to chloroplasts via
a secretory pathway. Subcellular fractionation of cauliflower cells
indicated the equivocal distribution of WSCP (data not shown). The
intracellular targeting function of the signal peptide of WSCP must be
tested experimentally and is currently under examination in a series of
transgenic studies in our laboratory.
Although the intracellular distribution of WSCP remains ambiguous, on
the other hand, the necessity of a water-soluble Chl carrier in the
chloroplast can be postulated as follows.
The photosynthetic apparatus is reorganized during acclimation to
various light environments (38, 39). Formation of photosynthetic Chl-protein complexes by redistribution of Chl has been reported (40-42); however, the detailed process of Chl redistribution has not
been clarified. It is conceivable that WSCP may play a role during this
process, because WSCP is able to extract Chl from thylakoid. However,
to support this hypothesis, it must be demonstrated that WSCP
transports Chl molecules into thylakoid pigment proteins.
When pea plants were subjected to drought stress (leaf water potential,
~ We thank Dr. Nikolai Lebedev
(University of Virginia, Charlottesville, VA) and Dr. Karlheinz Bortlik
(Nestle Research Center, Lausanne, Switzerland) for their helpful
comments. We are grateful to Dr. Yoshikazu Nishiguchi and Dr. Toshio
Imai (Department of Biology, Toho University) for their kind assistance
with the mass spectrometry. We were indebted to Professor Yoshiro
Kobayashi (Department of Biomolecular Science, Toho University) for
critical reading of the manuscript.
*
This work was supported in part by Grant-in-aid for
Scientific Research 10740376 (to H. S.) from the Ministry of
Education, Science and Culture, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB012699.
The abbreviations used are:
WSCP, water-soluble
chlorophyll protein; Chl, chlorophyll; PAGE, polyacrylamide gel
electrophoresis; MBP, maltose binding protein; RACE, rapid
amplification of cDNA ends; IPTG, isopropyl-1-thio-
Molecular Cloning and Functional Expression of a Water-soluble
Chlorophyll Protein, a Putative Carrier of Chlorophyll Molecules in
Cauliflower*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
Nucleotide and deduced amino acid sequences
from a cDNA encoding the WSCP from cauliflower.
Numbers of nucleotide, and deduced amino acid residues are
shown on the left and right sides of the
sequence, respectively. PCR primers to clone the cDNA for the
mature form WSCP and precursor of BnD22 (identical to WSCP, see text)
are double-underlined and underlined in the
figure, respectively. The vertical arrow indicates a
cleavage site for processing of the precursor. The cDNA encodes a
WSCP with 19 residues for the signal peptides and 199 residues for the
mature form of WSCP.
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Fig. 2.
Sequence alignment of the mature form of
cauliflower-WSCP and previously-reported drought-stress-related
proteins in Brassicaceae plants obtained from data bases. Residues
that matched that of the cauliflower WSCP are boxed.
Conserved amino acid residues among the six proteins listed are
indicated by asterisks. A motif for the Künitz
proteinase inhibitor family starting at Val 4 is indicated by the
thick bar. A homologous region to the (F/Y)DPLGL
motif is represented by the thin bar.
WSCP, deduced sequence of WSCP cDNA from B. oleracea determined in this study; BnD22,
drought-stress-induced 22-kDa protein in B. napus (7);
P22, drought-induced 22-kDa protein in R. sativus
(22); DIP, probable drought-induced protein in B. rapa (PIR accession no. S36621; GenBank accession no. Z25770);
WSCP, deduced sequence of WSCP cDNA from L. virginicum (DDBJ accession no. AB002589); AtDr4,
drought-suppressed protein in Arabidopsis thaliana
(24).
-D-galactopyranoside (IPTG)
to the bacterial culture medium induced a prominent polypeptide of 66 kDa (indicated by an arrow; compare lanes 1 and
2); this polypeptide represents a fusion of MBP (44 kDa) and
WSCP (22 kDa). Following sonication, much of the MBP-WSCP was found in
the soluble fraction of the cell extracts (compare lanes 3 and 4). The 66-kDa MBP-WSCP bound to an amylose column was
specifically eluted with 10 mM maltose (lane
5). Proteins expressed in pMAL-cRI can be cleaved with a proteinase, factor X
. The expressed MBP-WSCP was cleaved by the proteinase and analyzed by SDS-PAGE. The profile displayed a prominent 44-kDa band corresponding to MBP and several faint bands between 21 and
30 kDa (data not shown). Because we could not obtain a distinct band
for WSCP following factor-X digestion, we used the fusion protein to
analyze Chl binding.
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Fig. 3.
Expression and purification of the
recombinant MBP-WSCP expressed in E. coli. Lane
1, cell extract of uninduced E. coli; lane
2, cell extract of E. coli after inducing the
expression of MBP-WSCP with IPTG. Addition of IPTG induced a prominent
band corresponding to 66 kDa (indicated by an arrow) due to
a fusion protein of MBP (44 kDa) and WSCP (22 kDa); lane 3,
insoluble fraction of the E. coli broken with sonication;
lane 4, soluble fraction of sonicated E. coli;
lane 5, MBP-WSCP purified by amylose column
chromatography.
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Fig. 4.
Removal of Chls from thylakoid membranes and
structural change of MBP-WSCP as assayed by 8% PAGE. Five nmol of
MBP-WSCP was mixed with the thylakoid membrane preparation containing 0 (lane 1), 1.25 (lane 2), 5 (lane 3),
or 20 nmol (lane 4) of Chls. After 30 min of incubation, the
water-soluble fraction was recovered by centrifugation and subjected to
the 8% PAGE. Fluorescence of Chls was photographed on a long-waved UV
transilluminator (panel B). Protein bands were stained with
CBB (panel A). By binding of Chls, the monomer conformation
of MBP-WSCP converted mainly tetramer and minor hexamer conformations.
Only tetrameric and hexameric forms emitted fluorescence of Chls.
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Fig. 5.
Comparisons of absorption and those of fourth
derivative spectra of reconstituted MBP-WSCP and native WSCP purified
from cauliflower leaves. The thick line
represents a spectrum for native WSCP, and the dotted
line indicates that of MBP-WSCP. Panel A
indicates the absorption spectra of red and Soret bands. Panel
B displays the absorption and fourth derivative spectra of the red
band of both proteins.
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Fig. 6.
Comparisons of Chl forms in both native and
MBP-WSCP by curve-fitting analysis. Panel A
indicates a deconvolution profile of an absorption spectrum of native
WSCP, and panel B, that of MBP-WSCP. The analysis
deconvoluted both spectra into four major components assigned to be Chl
a peaked at 663, 671, 677, and 685 nm, and one component for
Chl b peaked at 653 nm. Although the intensities of the
component with a peaked at 677 nm were slightly different between the
two spectra, other components exhibited good agreement in both
intensity and wavelength between the two spectra.
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Fig. 7.
Comparisons of fluorescence spectra of
MBP-WSCP and native WSCP by curve-fitting analysis.
Panels A and B, emission spectra of
WSCPs excited Chl a at 440 nm; panels C and
D, emission spectra of WSCPs excited Chl b at 460 nm. Panels A and C, spectra of native WSCP;
panels B and D, spectra of MBP-WSCP. The
Chl-binding properties of MBP-WSCP and native WSCP are considered to be
identical.
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Fig. 8.
Conformational change of MBP-WSCP by binding
of purified Chls a and b. Zero, 12.5, 125, 250, or 500 pmol of Chl a (lanes 1-5) or Chl
b (lanes 6-10) were mixed in 20% ethanol with
the 500 pmol of the MBP-WSCP. Then the mixtures were separated on 8%
PAGE (detergent-free). Patterns of the proteins (panel A)
and Chls (panel B) were visualized. MBP-WSCP itself
possessed higher affinity to Chl a than that to Chl
b, based on the finding that mixing of Chl a and
MBP-WSCP at a 0.5:1 molar ratio exhibited no residual monomer, whereas
a 1:1 molar ratio of Chl b to MBP-WSCP still retained the
monomeric form.
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Fig. 9.
Hexamer formation of MBP-WSCP by mixing with
purified Chl a or thylakoid membranes. Patterns of
proteins (panel A) and Chls (panel B) were
displayed. Lane 1, no addition of Chls to MBP-WSCP;
lane 2, mixing of MBP-WSCP with purified Chl a at
a 0.75:1 molar ratio; lane 3, mixing of MBP-WSCP with Chls
in thylakoid membranes at a 0.75:4 molar ratio.
1, 3 rule" (31, 32) for
secretory proteins. These characteristics of the extension polypeptide
suggest that the WSCP precursor may be first transported into the
endoplasmic reticulum (ER). BnD22 was also predicted to be imported
into the ER (7). The fact that WSCP is a Chl-binding protein seems to conflict with the idea that WSCP could be transported into ER. A
hypothetical contact site of WSCP with Chl could be outside the
chloroplast. The various enzymes that increase during senescence and
seem likely to participate in chloroplast breakdown are not targeted to
the chloroplast (for review, see Ref. 33). Senescing chloroplasts exude
lipoidal blebs containing Chl or a similar fluorescing material. These
blebs could represent chloroplast components in transit to degradation
in the cytoplasm or vacuoles (33). As a different pathway of
chloroplast degradation, the removal of whole chloroplasts all at once,
as in phagocytosis, has been observed (33). In both degradative
pathways, Chl should be catabolized in the cytosol or vacuoles. If WSCP
is distributed in the ER or vacuoles, WSCP may participate in Chl
breakdown that occurs outside the chloroplasts.
1.3 MPa), Chl a declined by approximately 18% (43). Senescence (for review, see Ref. 44), as well as drought stress, may
cause Chl degradation. Although chlorophyllase, the enzyme catalyzing
the first step of the Chl degradation, is present in chloroplasts, its
activity is latent and observed only after detergent (45) or organic
solvent treatments (9). Recently, it has been shown that chlorophyllase
activity occurs on the inner chloroplastic envelope (23). Matile
et al. (23) hypothesized the existence of a water-soluble
carrier protein that transports Chls from thylakoid pigment-protein
complex to the inner chloroplastic envelope in senescent leaves (23,
44). WSCP is highly hydrophilic, may be located in chloroplasts (34),
is able to extract Chls from thylakoid pigment proteins (Fig. 4), and
is induced under stress conditions (3, 7, 8). All these features of
WSCP are in accord with the hypothesis that WSCP serves as a Chl carrier.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 81-474-72-7532;
Fax: 81-474-72-7532; E-mail: hsatoh{at}biomol.sci.toho-u.ac.jp.
-D-galactopyranoside; ER, endoplasmic
reticulum; PCR, polymerase chain reaction; CBB, Coomassie Brilliant Blue.
REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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