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INTRODUCTION |
In higher plants, lateral organs produced from the flanks of the
apical meristem exhibit defined adaxial-abaxial polarity. Recent
molecular genetic analysis has identified a number of factors involved
in establishing adaxial-abaxial organ polarity (1). These
investigations suggest that normal juxtaposition of factors regulating
abaxial and adaxial fate is necessary for normal organ growth and may
feed-back to support the maintenance of the apical meristem (2, 3).
The filamentous flower (fil) mutant was isolated
from Arabidopsis thaliana as one of the mutants that
have defects in flower development and morphogenesis (4). The
fil mutant forms two types of flowers. One is a flower with
an aberrant number and arrangement of organs, and the other is not a
shape of flower but a filament. It has been thought that the
FILAMENTOUS FLOWER (FIL)1 gene has a
role in the formation and development of floral meristems and
determination of the numbers of floral organs (5, 6). Recently, it is
proposed that the FIL gene is a member of a gene family
whose role appears to be pivotal in specifying the abaxial cell fate of
lateral organs (3). In addition to FIL, this gene family
consists of six members, including the flower specific genes
CRABS CLAW (CRC) (7), INNER NO OUTER
(INO) (8), and YABBY2 (YAB2),
YABBY3 (YAB3), and YABBY5
(YAB5) (3). YAB2, YAB3, and
FIL are expressed abaxially in all developing lateral organs
and are proposed to redundantly promote the abaxial fate of all lateral
organs in the plant (2, 3). The putative proteins encoded by this gene
family share amino-terminal zinc finger and carboxyl-terminal HMG-box
like domains (2, 3). The presence of these domains suggests that these
genes may function as transcriptional regulators. Consistent with this,
the FIL protein has been shown to contain a zinc ion and be localized
to the nucleus (2).
Recent studies have indicated that, in addition to DNA binding, zinc
fingers can work in protein·protein interactions supporting the formation of homo- and heterodimers as well as protein
self-association (9). This suggests the aspects that the protein
function of FIL and other members of this gene family may be mediated
through protein·protein interactions. Several sequence motifs have
been observed to accompany the zinc finger domain when they act in protein·protein interactions. These motifs include the
Kruppel-associated box (10), the poxvirus zinc finger domain (11), and
the SCAN domain (12). GATA family proteins are also capable of
protein·protein interactions (13). Nuclear bodies containing large
assemblies of GATA proteins have been observed by confocal microscopy
(14).
Among many zinc finger families, the LIM domain contains double zinc
finger structures that mediate specific contacts between proteins
participating in the formation of a multiprotein complex (15). The LIM
motif displays the consensus amino acid sequence Cys-X2-Cys-X16-23-His-X2-Cys-X2-Cys-X2-Cys-X16-23-Cys-X2-Cys (where X is any amino acid (16)), and spectroscopic studies of some LIM domains have revealed that they coordinate two zinc ions
(17-20). The RING finger domain contains eight conserved metal ligands
with similar spacing to those in the LIM motif (consensus motif
Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys -X2-Cys-X4-48-Cys-X2-Cys
(16)) and binds two zinc atoms per molecule in a cross-brace arrangement.
The FIL zinc finger domain occurs between positions 10 and 60 of the
FIL protein, near the amino terminus. In this study, we investigated
the FIL protein focusing on the zinc finger domain. We found that the
zinc finger motif of FIL,
Cys-X3-His-X5-His-X-Cys-X3-Cys -X2-Cys-X20-Cys-X-Cys-Cys,
is potentially an analogue of the LIM finger motif and that this domain
readily changes conformation and is involved in the self-association of
the FIL protein. FIL is thought to act as a transcriptional regulator
in the plant, but the molecular basis of its action has yet to be
demonstrated. We discuss the possibility that structural variability of
the FIL protein due to the changing conformation of the zinc finger domain may be central to its molecular and biological action.
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EXPERIMENTAL PROCEDURES |
Cells and Plasmids--
Plasmid pET28a and Escherichia
coli BL21(DE3) were from Novagen, Inc. Plasmid pGEX-2T was from
Amersham Pharmacia Biotech. Competent cells of E. coli JM109
(recA1, endA1, gyra96,
thi-1, hsdR17, supE44,
relA1,
(lac-proAB)/F[traD36,
proAB+, lacIq,
lacZ
M15]) for plasmid construction were from
Takara Shuzo Co., Ltd. Cells were grown in Luria-Bertani (LB) medium
(21) containing 50 mg/liter kanamycin or 100 mg/liter ampicillin.
Materials--
Restriction and DNA-modifying enzymes were from
Takara Shuzo Co., Ltd. The DNA ligation kit (ligation high) and KOD DNA
polymerase for the polymerase chain reaction (PCR) were from Toyobo
Co., Ltd. Isopropyl-
-D-thio-galactopyranoside (IPTG) was
from Wako Pure Chemical Industries, Ltd. The proteins used as standards for gel filtration were from Bio-Rad. A prestained protein marker for
Western blotting was from New England BioLabs. DNA oligomers were
synthesized by Sawady Technology Co., Ltd. The GelCode 6xHis protein
tag staining kit and tris(2-carboxyethyl)phosphine (TCEP-HCl) were from
Pierce. 4-(2-Pyridylazo)resorcinol (PAR) and
p-hydroxymercuriphenyl sulfonate (PMPS) were from
Sigma-Aldrich Fine Chemicals. Other chemicals were of reagent grade.
Construction of the Expression Vector--
The FIL
gene has been isolated as described previously (2). The gene was
amplified by PCR using the synthetic DNA oligomers P1
(5'-ATGTCGTCCATGGCCTCCCCTTCCTCAGCTG-3') and P2
(5'-TTTATCCTCGAGATAAGGAGTCACACCAACG-3') as primers.
Recognition sites of restriction endonucleases NcoI and
XhoI are shown by the two underlined
areas, respectively. Thirty cycles of PCR were performed
using a Trio-Thermoblock (Biometra) apparatus with KOD DNA polymerase
and using the procedures recommended by the supplier. The DNA product
was digested with NcoI and XhoI, and ligated to
the large NcoI-XhoI fragment of plasmid pET28a to
generate plasmid pETFIL, in which the FIL gene, encoding
FIL-(6-229) with a histidine tag at a carboxyl terminus, is
under the control of bacteriophage T7 transcription and translation
signals. Mutation of Cys56 to Ala was generated by overlap
PCR. Two initial PCRs were performed with primers P1 and P3
(5'-GATTGGTACAAGCACCACATCGG-3') and P2 and P4
(5'-CCGATGTGGTGCTTGTACCAATC-3'), where the substituted residues are underlined. The amplified DNA fragments were purified, and
a second PCR was carried out with primers P1 and P2. The sequences of
the wild-type and mutant FIL genes in pETFIL were confirmed by cycle sequencing using the ABI Prism dye terminator cycle sequencing ready reaction kits and ABI Prism 310 genetic analyzer from PerkinElmer Life Sciences. Truncation of the FIL gene was performed via
PCR with the synthetic DNA oligomers carrying BamHI and
EcoRI sites as primers, and using KOD DNA polymerase,
according the procedures recommended by the supplier. The DNA product
was digested with BamHI and EcoRI and ligated to
the large BamHI-EcoRI fragment of plasmid
pGEX-2T, thereby creating in-frame fusion with the glutathione
S-transferase (GST) gene. The nucleotide sequences of
the truncated FIL genes were confirmed as described above.
Overproduction of the FIL Protein--
Expression of the FIL
protein was induced in E. coli BL21(DE3) cells harboring the
plasmid pETFIL by the addition of IPTG in the presence of 10 µM zinc acetate. Cultivation of the E. coli transformants was carried out as described previously (2). Cells were
harvested by centrifugation and subjected to the purification procedures described below. The production of the FIL protein in cells
was examined by analyzing the whole-cell extract by sodium dodecyl
sulfate-PAGE (22). The solubility of FIL in cells was examined as
described previously (23).
Purification--
All purification procedures were carried out
at 4 °C. Cells from 1-liter culture were suspended in 50 ml of 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM
-mercaptoethanol, sonicated on ice for 3 min, and
centrifuged at 15,000 rpm (27,000 × g) for 30 min. EDTA (0.1 mM) was added to remove the zinc ions, which bind
nonspecifically to the protein. The resulting supernatant was dialyzed
against 20 mM sodium phosphate, pH 7.3, 5 mM
-mercaptoethanol at 4 °C for 4 h. The dialyzed solution was
applied to a HiTrap chelating column with Ni2+ ions from
Amersham Pharmacia Biotech. After the column was washed with buffer A
(20 mM Tris-HCl, pH 8.0, 0.1 M NaSCN, 5 mM
-mercaptoethanol, 0.6% sodium deoxycholate (DOC),
and 10% glycerol), the FIL protein was eluted with buffer A containing
100 mM imidazole. The eluant was applied to Superose 12 PC
3.2/30 in a SMART system from Amersham Pharmacia Biotech equilibrated
with buffer A. The elution of the FIL protein was performed with a flow
rate of 30 µl/min at 4 °C. The purity of the purified FIL protein
was analyzed by SDS-PAGE.
Zinc and Sulfhydryl Group Determination--
Zinc release
experiments were performed as described previously (24). The protein
solution was dialyzed against a buffer containing 10 mM
Tris-HCl, pH 8.0, 250 µM TCEP, 0.6% DOC, and 10%
glycerol to remove
-mercaptoethanol. To the mixture of 0.2 mM PMPS and 0.1 mM PAR in buffer B (30 mM Hepes-Na, pH 8.0, 10% glycerol, and 0.35 M
NaCl), the wild-type or mutant FIL protein was added to a final
concentration of 2 µM. Then, absorbance changes at 500 nm
were measured every 2 min. Zinc release from FIL was determined
indirectly by monitoring the formation of a
(PAR)2·Zn2+ complex (
500 = 66,000 cm
1 M
1) (25). Zinc
release in the presence of PAR only was also similarly analyzed by
measuring the absorbance change at 500 nm. Mercaptide bond formation
was directly followed at 250 nm.
Circular Dichroism--
CD spectra (200-260 nm) were measured
on a J-720 automatic spectropolarimeter (Japan Spectroscopic Co.,
Ltd.). Spectra were obtained using solutions containing the FIL
proteins at 0.3 mg/ml in 10 mM Tris-HCl, pH 8.5, containing
0.5 mM dithiothreitol. The EDTA-treated samples were
prepared by incubating the FIL protein solution with 10 mM
EDTA for 1 h at 20 °C, followed by the removal of excess EDTA
with a 20-h dialysis. The mean residue ellipticity [
], expressed
in units of deg·cm2·dmol
1, was calculated
by using an average amino acid molecular weight of 110. The helical
content of the protein was calculated by the method of Wu et
al. (26).
Protein Concentrations--
The protein concentration of FIL
with a tag was determined from the UV absorption at 280 nm. The
A
value of 0.62 for
FIL-(6-229) with a molecular weight of 26,305, was calculated by using
1,576 M
1·cm
1 for tyrosine
(7×) and 5,225 M
1·cm
1 for
tryptophan (1×) at 280 nm (27).
GST Pull-down Experiments with FIL-(6-229)--
GST-FIL fusion
proteins were prepared and adsorbed to the glutathione-agarose beads as
described previously (13). The beads containing the GST-FIL fusion
protein were incubated with the purified FIL-(6-229) by the
Ni2+ resin in 5-fold-diluted buffer A at 4 °C. After 7 days, the beads were collected and washed five times by the same
buffer. The bound material was then released by boiling in SDS loading
buffer and subjected to electrophoresis and Western blotting. The
presence of retained FIL-(6-229) with a histidine tag was detected
using a nickel-activated derivative of horseradish peroxidase (India HisProbe-HRP).
Secondary Structure Prediction and Homology Search--
The
secondary structure of FIL was predicted using a neural network system
that was offered as a service on the Predict Protein Server on the
Web. The program was provided by Rost and Sander (28). The
protein sequence of FIL was examined for similarity to known protein
sequences by BLASTP (29), and the sequence alignment was performed by
ClustalW (30). Both were available on the Web.
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RESULTS |
Overproduction and Purification--
Expression of the
FIL gene in the plasmid pETFIL was induced in E. coli by the addition of IPTG as described previously (2). The
cultivation of the transformants was performed at 30 °C to obtain
the FIL protein in soluble form. Five amino acid residues at the amino
terminus of FIL were removed to avoid the heterogeneity of the
expressed protein. FIL-(6-229) was efficiently and selectively trapped
by the chelating column when a histidine tag was attached to the
carboxyl terminus. Elution with buffer A (20 mM Tris-HCl, pH 8.0, 0.1 M NaSCN, 5 mM
-mercaptoethanol,
0.6% DOC, and 10% glycerol) containing 100 mM imidazole
yielded 2.0 mg of protein product containing FIL from 1 liter of
culture, which was identified as a single band using SDS-PAGE analysis
(data not shown).
Self-assembly and Dissociation of the FIL Protein--
When the
FIL protein purification was performed in the absence of solubilizers
and dissociating agents, the FIL protein formed a precipitate. This
phenomenon prevented both the characterization and further increase in
the concentration of the FIL protein. Changing pH and the addition of
glycerol, Triton X-100, octylglycoside, CHAPS, and NaCl failed to
prevent the formation of this precipitate, but the addition of 0.55 M arginine and 0.44 M sucrose did (31). Therefore, gel filtration of the FIL protein using Superose 12 was
performed at pH 5.5 in the presence of arginine and sucrose. The
elution profile showed that all the FIL protein formed a multimer, which was eluted out with a void volume, and the peak corresponding to
the FIL monomer was not observed (data not shown). This result suggests
that more than twenty molecules of FIL assemble together as a multimer
and cause FIL protein precipitation in the absence of the solubilizer.
Among several dissociating agents examined to dissociate the multimer
of the FIL protein, DOC (32) and sodium thiocyanate (33) were most
effective. Therefore, gel filtration was performed at pH 8.0 in the
presence of 0.6% DOC and 0.1 M sodium thiocyanate. The
elution profile showed two peaks that corresponded to the multimer
eluted with the void volume (0.9 ml), and the FIL monomer protein
eluted with a retention volume of 1.25 ml (Fig.
1a). Sedimentation equilibrium
experiments were carried out to determine the molecular mass of this
protein. Concentration versus radial distance profiles were
obtained at three different rotor speeds (data not shown), and a
nonlinear least squares analysis revealed that the data fit a simple
single species model exhibiting a slight tendency for aggregation and a
molecular mass of 27,100 ± 240 Da. This is very close to the
theoretical mass of 26,305. When the FIL solution was applied to the
column for gel filtration, immediately after recovery from the
chelating column, the elution profile showed that the multimer and
monomer forms of the FIL protein constituted 27 and 73% of the FIL
protein, respectively (Fig. 1b).

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Fig. 1.
The elution profiles of the gel filtration
chromatography of the wild-type FIL protein and the time courses of the
amount of the FIL monomer and multimer in the absence or presence of
EDTA. a, the solution containing the wild-type FIL
protein in the absence of EDTA was applied immediately after the
elution from the Ni2+ resin (thin line) or
applied after standing at 4 °C for 6 days (thick line) to
Superose 12 PC 3.2/30 in a SMART system from Amersham Pharmacia Biotech
equilibrated with buffer A. The elution of the FIL protein was
performed with a flow rate of 30 µl/min at 4 °C. The retention
volume of the FIL multimer was 0.9 ml, and that of the FIL monomer was
1.25 ml. b, the relative amounts of the FIL multimer ( )
and the FIL monomer ( ) in the wild-type FIL solution kept at 4 °C
in the absence of EDTA were monitored for 6 days by comparing the areas
of the peaks corresponding to the multimer and the monomer in the
elution profile. c, EDTA (10 mM) was added to
the solution containing the wild-type FIL protein. It was applied
immediately (thin line) or after standing at 4 °C for 6 days (thick line) to the same column. d, the
relative amounts of the FIL multimer ( ) and the FIL monomer ( ) in
the wild-type FIL solution kept at 4 °C in the presence of 10 mM EDTA were monitored for 6 days as described above.
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A time course for the relative proportions of the FIL multimer and
monomer forms were examined when the FIL protein with a total
concentration of 2.0 mg/ml was kept in buffer A at pH 8.0 and 4 °C
for 6 days. Fig. 1b shows the decrease of the monomer and
the increase of the multimer forms of the FIL protein observed over the
6-day period. The shift from the FIL monomer to the multimer form
occurred gradually over this time. After 6 days, the monomer had
decreased to 67% from 73%, conversely, the multimer increased to 33%
from 27% (Fig. 1b). After 30 days, the relative amount of
the multimer was ~70% and that of the monomer was ~30% (data not
shown). Lowering the pH of the buffer increased the multimerization of
FIL, whereas dilution of the FIL solution diminished the
multimerization (data not shown).
The addition of EDTA to the FIL solution markedly enhanced the
multimerization of FIL protein (Fig. 1, c and d).
When the relative decrease of the monomer and the increase of the
multimer in the presence of EDTA at 4 °C was monitored over 6 days
(Fig. 1d), the percentage of FIL monomer protein had
decreased to 48% from 73% by the end of this period. Conversely, the
percentage of FIL multimer protein had increased to 52% from 27%
(Fig. 1d).
These results suggest that the formation of the FIL protein multimer
could be related to a spontaneous release of a zinc ion from the FIL
protein and that the addition of EDTA may enhance this process. We
decided to examine the theoretical structure of the zinc finger domain
of FIL to help investigate this possibility.
Comparison of the Amino Acid Sequences of the FIL, YAB2, and YAB3
Proteins--
The FIL protein has the amino-terminal C-rich structure
with a
CX3HX5HXCX3CX2CX20CXCC
(CH2C6) motif between positions 14 and 57 and the
carboxyl-terminal HMG-box like domain for the potential DNA binding.
The deduced amino acid sequence of the FIL protein was compared with
those of the YAB2 and YAB3 proteins. Fig.
2 shows the alignment of the three
sequences. The zinc finger domain of the FIL protein has seven cysteine
residues at positions 14, 26, 30, 33, 54, 56, and 57, and two histidine
residues at positions 18 and 24 (CH2C6 motif) (Fig. 2). The zinc finger
domain of the YAB2 protein has five cysteine residues at positions 11, 15, 18, 39, and 42, and two histidine residues at positions 14 and 41 (CHC3HC motif) (Fig. 2). The zinc finger domain of the YAB3 protein has
five cysteine residues at positions 26, 30, 33, 54, and 57, and three
histidine residues at positions 18, 29, and 56 (HCHC3HC motif) (Fig.
2). The five cysteine residues at positions 26, 30, 33, 54, and 57 of
the FIL protein are conserved in the three sequences (Fig. 2), and four
cysteine residues at positions 30, 33, 54, and 57 are thought to be in
the canonical positions for the zinc ligation. However, the cysteine
residues at positions 14 and 56 of FIL are not conserved among the
sequences of the three zinc finger domains shown in Fig. 2. Therefore,
Cys56 was considered as a candidate residue for
facilitating the observed zinc release from the FIL protein. To examine
a possible role of the Cys56 in zinc ligation
destabilization, alanine was substituted for cysteine at position 56 in
the FIL protein.

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Fig. 2.
Sequence alignment of the FIL, YAB2, and YAB3
proteins. The alignment of the deduced amino acid sequences of the
FIL, YAB2, and YAB3 proteins was performed by ClustalW (30). The zinc
finger domains of the FIL, YAB2, and YAB3 proteins are
shaded. The five conserved cysteine residues among the three
sequences in the zinc finger domains are indicated by
asterisks. All cysteine and histidine residues in the three
zinc finger domains are underlined. The cysteine residue at
position 56 in the zinc finger domain of FIL is indicated by an
arrow. The region between positions 45 and 107 of the FIL
protein, which mediates the FIL·FIL interaction, is indicated by
double underlining. The regions that form five putative
-helices in the FIL protein suggested by the secondary structure
prediction and the corresponding regions conserved in the YAB2 and YAB3
proteins are indicated by rectangles.
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Substitution of Alanine for Cysteine at Position 56 Suppressed the
Multimerization of the Protein and Zinc Release from the Zinc Finger
Domain--
To examine a possible role of the Cys56 in
zinc ligation destabilization, alanine was substituted for cysteine at
position 56. The solution of the C56A mutant protein was analyzed by
gel filtration chromatography (Fig.
3a). Immediately after the
elution from the Ni2+ resin, the approximate percentage of
the fil C56A multimer was 10% compared with 80% for the fil C56A
monomer and 10% for the fil C56A oligomer (Fig. 3b). This
fil C56A oligomer was eluted with a retention volume of 1.09 ml, which
was 0.16 ml smaller than that of the fil C56A monomer (1.25 ml) and
0.19 ml larger than that of the fil C56A multimer (0.9 ml) (Fig.
3a). The relative amount of the fil C56A multimer remained
at 10% for 6 days (Fig. 3b). These results differed
markedly from comparable experiments with the wild-type FIL protein
(Fig. 1b), suggesting that the multimerization of the FIL
protein is effectively suppressed by the substitution of Ala for
Cys56. On the other hand, the monomer of fil C56A gradually
decreased, with a commensurate, gradual increase in the concentration
of the fil C56A oligomer over 6 days (Fig. 3b). The
formation of an oligomer was not observed in the elution profile of the
wild-type protein (Fig. 1a).

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Fig. 3.
The elution profiles of the gel filtration
chromatography of the C56A mutant protein and the time courses of the
amount of the C56A monomer, oligomer, and multimer in the absence or
presence of EDTA. a, the solution containing the C56A
mutant protein in the absence of EDTA was applied immediately after the
elution from the Ni2+ resin (thin line) or
applied after standing at 4 °C for 6 days (thick line) to
the column as described in the legend of Fig. 1. The retention volume
of the C56A multimer was 0.9 ml, that of the C56A oligomer was 1.09, and that of the C56A monomer was 1.25 ml. b, the relative
amounts of the multimer ( ), that of the oligomer (x) and
that of the monomer ( ) in the C56A solution kept at 4 °C in the
absence of EDTA were monitored for 6 days as described. c,
the solution containing the C56A protein in the presence of EDTA was
applied immediately after the addition of EDTA (thin line)
or after standing at 4 °C for 6 days (thick line) to the
same column. d, the relative amounts of the multimer ( ),
that of the oligomer (x), and that of the monomer ( ) in
the C56A protein solution kept at 4 °C in the presence of 10 mM EDTA were monitored for 6 days as described above.
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As was observed for wild-type FIL protein, the addition of EDTA
increased the amount of fil C56A multimer but the effect was less
(Figs. 1c, 1d, 3c, and 3d).
Moreover, it seems that the formation of the fil C56A oligomer was not
influenced by EDTA (Fig. 3, b and d). This
suggests that the fil C56A oligomer forms irrespective of whether there
is zinc release from the zinc finger domain.
Structural Change of FIL Caused by the Addition of EDTA--
Fig.
4 shows the far-UV CD spectra of the
wild-type FIL and fil C56A proteins at pH 8.5 and 20 °C. Both the
spectra (Fig. 4, a and b) are very similar to
each other, and the helical content of the protein was 14.3% for the
wild-type protein, and 14.9% for the C56A mutant. The addition of 10 mM EDTA to both the wild-type FIL and fil C56A mutant
proteins produced CD spectral changes and a decrease in the helical
content. (12.0% for the wild-type protein, and 13.1% for the fil C56A
protein). Notably, the decrease in the helical content of the wild-type
(2.3%) was larger than that of C56A (1.8%), indicating that EDTA
effected a greater structural change on the wild-type FIL protein than
on the fil C56A mutant protein. These results suggest that the addition
of EDTA may interfere with zinc ligation by the zinc finger in the
wild-type FIL protein causing zinc release. Under this proposal the
loss of zinc then results in structural changes in the FIL protein that
increases its tendency for self-association and multimerization. To
investigate the potential role for zinc in this process we induced and
monitored zinc loss from the wild-type FIL and fil C56A mutant
proteins.

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Fig. 4.
CD spectra of the wild-type FIL and C56A
mutant fil proteins. a, the far-UV CD spectrum of the
wild-type FIL protein solution is shown by a thin solid line
and that of the EDTA-treated FIL protein solution is shown by a
thick solid line. b, the far-UV CD spectrum of
the C56A mutant protein solution is shown by a thin solid
line and that of the EDTA-treated C56A protein solution is shown
by a thick solid line. The measurement was performed as
described under "Experimental Procedures."
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Release of Zinc Ions from FIL--
The release of zinc ions from
FIL was induced by chemical modification using PMPS and was monitored
in the presence of the metallochromic indicator PAR (24). All zinc ions
released from FIL were converted to the highly absorbant
(PAR)2·Zn2+ complex (
= 6.6 × 104 M
1 cm
1 at
500 nm) (25), which was determined by measuring absorbance changes at
500 nm. As shown in Fig. 5a,
the mixing of the wild-type or fil C56A mutant protein (2 µM) with PMPS (0.2 mM) in the presence of PAR
(0.1 mM) resulted in the increase of absorbance at 500 nm
for 10 min and reached plateau values of 0.25 (
A500). This value corresponds to the
absorbance of 3.8 µM
(PAR)2·Zn2+ complex solution and is
equivalent to the release of 1.9 zinc ions per protein (the
experimental value of 3.8 µM approximates the theoretical
value of 4.0 µM for two zinc ions per protein assuming
the oxidation of a few cysteine residues during the protein preparation). The mercaptide bond formation between PMPS and FIL cysteine residues was monitored at 250 nm in the absence of PAR. The
absorbance change over the 20 min was 0.21 for the wild-type protein
and 0.18 for the fil C56A mutant (Fig. 5b). These results are consistent with there being seven cysteine residues per FIL protein and six cysteine residues per fil C56A mutant protein as
modified by PMPS (based on the assumption that 95% of sulfhydryl group
was active in the cysteine modification).

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Fig. 5.
Determination of the zinc and sulfhydryl
group content of the wild-type FIL and C56A mutant fil proteins.
a, zinc determination. The absorbance change at 500 nm was
monitored for 20 min after the addition of the wild-type FIL protein (2 µM) ( ) or the C56A mutant protein (2 µM)
( ) to buffer B containing 0.2 mM PMPS and 0.1 mM PAR. b, sulfhydryl group determination. The
absorbance change at 250 nm was monitored for 20 min after the addition
of the wild-type FIL protein (2 µM) ( ) or the C56A
mutant protein (2 µM) ( ) to buffer B containing 0.2 mM PMPS in the absence of PAR. c, zinc release
from the wild-type FIL or C56A mutant fil protein without cysteine
modification. The absorbance change at 500 nm was monitored for 8 h after the addition of the wild-type FIL protein (2 µM)
( ) or the C56A mutant protein (2 µM) ( ) to
buffer B containing 0.1 mM PAR in the absence of
PMPS.
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The zinc release was observed from both the wild-type FIL or fil C56A
mutant proteins in the presence of PAR alone without any cysteine
modification (Fig. 5c). The absorbance change at 500 nm for
8 h was 0.13, indicating that almost one zinc ion was released per
protein from both the wild-type and C56A mutant proteins during this
period (Fig. 5c). These results suggest that, in the presence of PAR alone, only one of the two zinc ions in the FIL wild-type and fil C56A mutant protein can be released. At the same
time, the rate of zinc release from the wild-type protein seemed to be
about one order faster than that from the fil C56A protein (Fig.
5c). Thus the wild-type cysteine residue at position 56 appears to enhance the speed at which the zinc ion is released from the
wild-type FIL protein.
Spontaneous zinc release from the wild-type FIL protein (2 mg/ml in
buffer A) in the absence of PAR and PMPS was also detected when the FIL
protein was separated from the free zinc ions by dialysis. In this case
the zinc release reached a plateau for 7 days at 4 °C (data not shown).
Determination of the Region Involved in FIL
Self-association--
The FIL protein contains two zinc ions, and one
zinc ion is released more easily from the FIL protein than the other
(Fig. 5). The loss of the zinc ion likely causes the structural change of the zinc finger domain illustrated in Fig. 4, which is linked to
increased self-association by the FIL protein (Fig. 1). To determine
the region that is involved in the self-association of FIL,
FIL-(6-229) was mixed with glutathione-agarose beads coated with
either GST, GST-FIL1-45, GST-FIL1-75, GST-FIL1-114, GST-FIL65-229, GST-FIL107-229, or GST-FIL1-229 (Fig.
6a). After extensive washing, the protein retained on the beads was subjected to electrophoresis (Fig. 6b) and Western blotting (Fig. 6c). As
shown in Fig. 6c, the GST-FIL1-75, GST-FIL1-114,
GST-FIL65-229, and GST-FIL1-229 fusions could retain FIL-(6-229). In
contrast, the beads coated with GST, GST-FIL1-45, and GST-FIL107-229
could not retain it. These results imply that the residues between
positions 45 and 107 contribute to FIL self-association (Fig.
6a), and the residues between positions 45 and 60 and those
between positions 60 and 100 are specifically involved. The former
region corresponds to the carboxyl half of the zinc finger domain, and
the latter corresponds to the adjacent hydrophobic region containing
two putative
-helices.

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Fig. 6.
Determination of the region that mediates the
association of the FIL protein. a, diagrams of a
GST protein and six GST-FIL fusion protein forms used for the analysis.
Open rectangles indicate the GST proteins, and black
rectangles indicate the truncated and full-length FIL proteins.
The region indicated by gray shading (positions between 45 and 107) is that mediating the protein·protein interaction.
b, SDS-PAGE. GST-FIL fusion proteins and GST revealed by
Coomassie Blue staining after Western blotting are shown. c,
Western blot. The bands of FIL-(6-229), with histidine tag retained by
GST-FIL1-229, GST-FIL65-229, GST-FIL1-114, and GST-FIL1-75 revealed
by nickel-activated derivative of horseradish peroxidase (India
HisProbe-HRP), are shown.
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Secondary Structure Prediction of FIL--
The analysis to predict
the secondary structure of the FIL protein suggested that residues at
positions 74-80, 89-95, 152-157, 169-179, and 204-214 had the
propensity to form
-helices (helices I,
II, III, IV, and V in Fig.
2), whereas residues at positions 25-30, 34-40, and 45-54 in the
zinc finger domain had the propensity to form
-sheet structures
(data not shown). Of the five putative
-helices, helices
I and II (Fig. 2) contain five leucine residues in the
hydrophobic region adjacent to the zinc finger domain, whereas
III, IV, and V in the HMG-box-like
domain of FIL (Fig. 2) have no leucine residues. It is assumed that
helices I and II are likely to participate in the
association of proteins by way of the leucine·leucine interactions.
These two helices (I and II) of FIL are not
conserved in the sequences of the YAB2 and YAB3 proteins. Although,
among the three helices in the carboxyl-terminal HMG-box-like domain of
FIL, helices III and IV are conserved in the FIL,
YAB2, and YAB3 proteins (Fig. 2).
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DISCUSSION |
Molecular genetic analysis indicates that FIL has an
important role in specifying abaxial cell fate in the apical and floral meristems (2-6). Little is known, however, about the in
vivo action of the FIL protein except that it is likely to be a
transcriptional regulator. In this study we have investigated the role
of zinc ions in the FIL zinc finger domain and the structural
organization of the protein to understand the functional properties of
the FIL protein.
It is proposed that FIL is a member of the gene family
including CRC (7), INO (8), YAB2,
YAB3, and YAB5 (3). Among them, FIL,
YAB2, and YAB3 are thought to be highly
correlated with each other to specify the abaxial cell fate. The
putative proteins encoded by these genes have a zinc finger domain
toward the amino terminus and the HMG-box-like domain toward the
carboxyl terminus. The YAB2 protein has a CHC3HC zinc finger motif, and the YAB3 protein has a HCHC3HC motif (Fig. 2) based on the putative amino acid sequences reported by Siegfried et al. (3). It
has been predicted that theYAB2 and YAB3 proteins have a
Cys2Cys2 zinc-finger domain and bind a zinc ion
per protein. On the other hand, our results show that the CH2C6 zinc
finger motif of FIL binds two zinc ions per protein. It is possible
that the YAB2 and YAB3 proteins bind two metal ions? It seems to be
difficult for the zinc finger domain of YAB2 to bind two zinc ions.
However, the two-metal binding by the YAB3 protein might be possible,
if the seven potential zinc coordinating residues (His18,
Cys26, His29, Cys30,
Cys33, Cys54, and Cys57) of the
YAB3 protein are arranged around two zinc ions as reported on the
crystal structure of the RAG1 dimerization domain, in which two zinc
ions share one cysteine residue in the binding site (34). It might be
expected that the FIL, YAB2, and YAB3 proteins share in the in
vivo function of the different zinc finger structures.
The arrangement of the cysteine and histidine residues of the zinc
finger domain of FIL, particularly in the carboxyl-terminal half of the
zinc finger domain, has some similarities to that of the LIM finger
domain. Homology search using BLASTP (29) exhibited the 33% identity
between positions 26-67 of FIL and the positions 583-662 of LimA from
Dictyostelium discoideum, although the position and the
number of histidine residues in the amino-terminal-half of the zinc
finger domain of FIL are inconsistent with that of the LIM finger
domain. It was confirmed that the fusion protein GST-FIL1-45 binds a
zinc ion (data not shown). This result strongly suggests that the
amino-terminal half of the zinc finger domain has an ability to bind a
zinc ion. According to the arrangement of the LIM finger motif (16), it
might be assumed that Cys14, His18,
His24, and Cys26 are the zinc coordinating
residues of what could be classified as site 1, and Cys30,
Cys33, Cys54, and Cys57 are those
of site 2 in the zinc finger domain of the FIL protein (Fig. 2).
Our results strongly suggest that the cysteine residue at position 56 contributes to the weakening of the ligation of one of the zinc ions in
the zinc finger domain of FIL. Significantly, extra cysteine residues
like Cys56 of FIL are observed in other zinc finger domains
such as the penultimate cysteine residue of the RING finger domain of
COP1 (35) and the second cysteine residue of the first RING finger domain of PRT1 from Arabidopsis (36). Based on our
results, it seems possible that these extra cysteine residues may
disturb the zinc ligation by the zinc-coordinating residues at the
canonical positions.
The slow but spontaneous release of the zinc ion from the zinc finger
domain of FIL results in a structural change to the FIL protein.
Presumably, this change in FIL structure exposes the adhesive surface
of the FIL protein and allows for the formation of FIL multimers (Fig.
1, a and c). Although there is no evidence that
the formation of the fil C56A protein oligomers shown in Fig. 3
(a and c) are related to the loss of the zinc
ion, their formation may reflect a similar but reduced tendency for
self-association observed for the wild-type FIL protein. Zinc release
and the theoretical exposure of the adhesive surface that results may
be partially suppressed in the fil C56A mutant, preventing multimer
formation but allowing oligomer formation (Fig. 3b).
It seems that the structure of the zinc finger domain of FIL is
inherently unstable. This conformational flexibility may allow the
rapid disassociation of FIL protein from the transcriptional machinery.
Alternatively, the multimerization of the FIL protein that occurs after
loss of a zinc ion in vitro, may (if it occurs in
vivo) increase the potency of FIL as a transcription factor. This
is supported by preliminary results indicating that FIL can also
self-associate in the DNA-bound form (data not shown). There are also
some examples of proteins that increase their local concentration and
efficacy by self-association. The Ikaros protein forms a cluster in the
association with transcriptionally silent genes to act as a recruiter
(37). Formation of the multimer of the Sp1 protein at the DNA loop
juncture leads to an increase in the concentration of activator protein
at the promoter (38). Thus the HMG domain of FIL may bind DNA, and the
self-association could increase the efficacy of FIL as a
transcriptional regulator.
The erythroid protein GATA-1 contains two zinc fingers (13). The
C-finger is for sequence-specific DNA binding, and the N-finger is both
for the modulation of DNA binding and for the protein·protein
interactions. Deletion analysis performed on the GATA-1 protein to
identify the region for the protein interaction has shown that a
25-residue region at the carboxyl-terminal half of the N-finger domain
is sufficient for GATA-1 protein·protein interaction (13). Deletion
analysis on the FIL protein in this study indicates that the region
between positions 45 and 107 participates in the FIL·FIL interactions
(Fig. 6). This region corresponds to the carboxyl-terminal half of the
proposed zinc finger domain and a following hydrophobic region
containing two putative
-helices. The region contains nine leucine
residues, with the two putative
-helices containing five of the nine
(Fig. 2). Many transcription factors contain extended motifs that
mediate oligomerization to presumably create an active complex. These
motifs include Kruppel-associated box (10), poxvirus zinc finger
domains (11), and the SCAN domain, which, like the
-helices region
of FIL, is leucine-rich (the 80-amino acid region of the SCAN-box
contains 14 conserved leucine residues) (12). Notably, these conserved
leucine residues are thought to play an important role in the
oligomerization and self-association properties reported for the
SCAN-box (12). These data reinforce the possibility that the FIL
leucine residues between positions 45 and 107 (Fig. 2) play an
important role in protein·protein interactions.
Both the YAB2 and YAB3 proteins have similar hydrophobic regions
abounding with leucine residues adjacent to their zinc finger domains (Fig. 2). Because FIL, YAB2, and
YAB3 have similar patterns of expression in
Arabidopsis (3), heteromolecular interaction between the FIL
and YAB2 or YAB3 proteins might be possible. Because the sequence of
the region between positions 45 and 107 of FIL has 18 and 37%
identities to those of the corresponding regions of YAB2 and YAB3
proteins, respectively (Fig. 2), the possibility of the heteromolecular
interaction between FIL and YAB3 might be higher than that between FIL
and YAB2. However, the two
-helices (I and II
in Fig. 2) predicted in the FIL protein were not conserved in the
corresponding regions of the YAB2 and YAB3 proteins (Fig. 2).
In summary, the zinc finger domain of FIL was found to bind two zinc
ions per protein. One of the two zinc ions is released spontaneously
and is followed by the self-assembly of the FIL protein. The cysteine
residue at position 56 is one factor that promotes rapid zinc release
from the FIL protein. The hydrophobic leucine-rich region between
positions 45 and 107 mediates the self-association of the FIL protein.