Download The Calcium-Binding Activity of a Vacuole

The Calcium-Binding Activity of a Vacuole-Associated,
Dehydrin-Like Protein Is Regulated by Phosphorylation1
Bruce J. Heyen2, Muath K. Alsheikh, Elizabeth A. Smith, Carl F. Torvik, Darren F. Seals3, and
Stephen K. Randall*
Department of Biology, Indiana University-Purdue University at Indianapolis, 723 West Michigan Street,
Indianapolis, Indiana 46202–5132
A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium
graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles.
Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity
with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties
similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having
an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of
flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 ⫾ 0.142 mm and 0.64 ⫾ 0.08 mm,
and has an estimated 24.7 ⫾ 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are
modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated
protein. VCaB45 is an “in vitro” substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting
in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45
suggest potential functions.
The vacuole is a reservoir for calcium (Machlon,
1984) and consequently plays an important role in
calcium homeostasis (Miller et al., 1990; Allen and
Sanders, 1995; Sanders et al., 1999). Regulation of
vacuole calcium levels is complex involving a variety
of calcium channels and pumps (Sanders et al., 1999;
Sze et al., 2000). Sustained elevated levels of cytosolic
calcium can be toxic (Hepler and Wayne, 1985), so
under normal conditions, cytosolic calcium levels increase only transiently. Proteinaceous calcium buffers may serve as homeostats to attenuate the signal
transduction system. Well-characterized protein calcium buffers include calreticulin and calsequestrin
(Ostwald and MacLennon, 1974; Campbell et al.,
1983b). Homologs of calsequestrin (Krause et al.,
1989; Xing et al., 1994), calreticulin (Chen et al., 1994;
Napier et al., 1995; Nelson et al., 1997), and calnexin
(Li et al., 1998) have been identified in plants. These
calcium-binding proteins can bind on the order of 20
to 50 calcium ions with both high- (1–3 sites per
protein) and low- (20–50 sites per protein) affinity
1
This work was supported in part by Purdue University (Research Fellowship) and by the U.S. Department of AgricultureNational Research Initiative Competitive Grants Program (grant
no. 99 –35100 –7668 to S.K.R.).
2
Present address: Departments of Biology and Chemistry, Tabor College, 400 South Jefferson, Hillsboro, KS 67063.
3
Present address: Van Andel Institute, 333 Bostwick Avenue
NE, Grand Rapids, MI 49503.
* Corresponding author; e-mail [email protected]; fax 317–274 –
2846.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.002550.
sites. The levels of calcium binding proteins may
have a significant impact on signaling processes and
may regulate second messenger transmission (Camacho and Lechleiter, 1995; Mery et al., 1996; Coppolino
et al., 1997). In an alternative role, calcium-dependent
interactions of calnexin and calreticulin have been
characterized with a variety of proteins (Nigam et al.,
1994; Peterson et al., 1995) and both are implicated in
the promotion of correct protein folding (Hebert et
al., 1996). These latter activities clearly suggest a
molecular chaperone role. Recently, a high-capacity,
low-affinity calcium-binding protein was localized to
the radish (Raphanus sativus) root vacuole. This protein did not show sequence resemblance to EF handcontaining calcium-binding proteins, calsequestrin,
or dehydrins (Yuasa and Maeshima, 2000).
Most members of the dehydrin superfamily of related genes are expressed during periods of low water
content or during exposure to environmental stresses
where osmotic stress is a component of the stress
mechanism (Skriver and Mundy, 1990; Close et al.,
1993b; Close, 1997). Included in the dehydrin superfamily are several late embryogenesis-abundant
mRNA-encoded proteins (LEAs), defined by the presence of the K domain (the prototypical sequence being
EKKGIMDKIKEKLPG). Many dehydrins additionally
contain an S domain (a tract of six–seven Sers) upstream of the amino-terminal-most K domain. Dehydrins have potential phosphorylation sites and can be
phosphorylated (Vilardel et al., 1990; Plana et al., 1991;
C.F. Torvik and S.K. Randall, unpublished data). Despite the progress in understanding the gene regulation affecting expression of the dehydrin proteins, the
Downloaded
from on www.plantphysiol.org
June 15, 2017 - Published
by www.plantphysiol.org
Plant Physiology, October 2002, Vol.
130, pp. 675–687,
© 2002
American Society of Plant Biologists
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
675
Heyen et al.
biochemical function of these proteins and their role in
cryoprotection have remained speculative.
Calcium ligand blots, a valuable technique for the
identification of novel calcium-binding proteins
(Campbell et al., 1983a, 1983b; Maruyama and Nonomura, 1984), specifically identifies a variety of
calcium-binding proteins including the EF handcontaining calmodulin (Chen et al., 1994), the 14-3-3
calcium-binding putative transcription factor (Lu et
al., 1994), and the calcium buffers calreticulin (Chen
et al., 1994) and calsequestrin (Franceschi et al., 1993).
Calcium ligand blots revealed only a limited number
of calcium-binding proteins (four–five) associated
with plant membranes (Randall, 1992). In this report,
we characterize one of these proteins, the vacuolar
calcium-binding protein known as VCaB45. VCaB45
is likely a member of the dehydrin family of proteins.
These findings suggest that calcium binding could be
a general property of dehydrins and suggest potential functions of dehydrins as calcium buffers or perhaps as calcium-dependent chaperones. We further
demonstrate that the calcium-binding activity of
VCaB45 is dependent upon its phosphorylation
status.
RESULTS
Purification of VCaB45
The presence of a 45-kD calcium-binding protein in
vacuole-enriched membrane fractions was noted previously (Randall, 1992). This report describes the purification, identification, and characterization of this
protein. The 45-kD protein was purified from a
vacuole-enriched membrane fraction, first by permeabilizing membrane vesicles with 0.2% (w/w) Triton
X-100, resulting in a fraction highly enriched in vacuole lumenal proteins (see methods). These proteins
were further fractionated by anion-exchange chromatography (Randall, 1992). Peak fractions were resolved on two-dimensional gels. Figure 1 shows a
typical two-dimensional gel separation of the 0.2%
(w/w) Triton X-100 extract of vacuole membranes;
analyzed by protein and by 45Ca ligand blots. At all
purification stages, VCaB45 was identified by the
45
Ca ligand-blotting method. The final purified protein had an apparent molecular mass of 45 kD and an
observed pI of 5.2 ⫾ 0.2 (average of five independent
determinations). Although VCaB45 represents a significant portion of the 0.2% (w/w) Triton X-100soluble vacuole proteins, it is likely not a major vacuole protein constituent (Randall, 1992; based upon
the purification, see discussion in Fig. 2A).
Antibody was raised to the purified protein in mice
by injection of spots of protein excised from blots of
several two-dimensional gels. The enrichment of
VCaB45 through the various purification stages is illustrated by using this antibody (Fig. 2A). A semiquantitative estimate of purification based upon the
western blot indicated a 400- to 500-fold purification
676
Figure 1. Two-dimensional analysis of 0.2% (w/w) Triton X-100extracted vacuole membranes. After the Triton X-100 treatment, the
soluble phase was separated first by isoelectric focusing and then by
SDS-PAGE (O’ Farrell, 1975). The pH gradient was established in the
first dimension with 0.5% (w/v) ampholytes (3.5–5 pH range), 0.75%
(w/v) ampholytes (4–6 pH range), and 0.75% ampholytes (5–7 pH
range). After electrophoresis and transfer to nitrocellulose, the blot
was first probed with 45calcium (B, as described) and later stained
with Ponceau S to detect protein (A). The arrows in A indicate the
protein spots that correspond to the calcium-binding activity detected in B. The white circles in B indicate calcium-binding activity.
The pH values were obtained by slicing a parallel first dimension gel,
incubating the slices in deionized water, and then measuring the pH
with an pH meter. The average value of VCaB45 was deduced to be
5.2 ⫾ 0.2 (average of five determinations). Molecular mass standards
are indicated on the left.
(from initial homogenate to anion-exchange peak fraction). The enrichment of VCaB45 in total membranes
compared with total cellular extract (approximately
10-fold) is indicative of a predominantly membrane/
organelle localization for VCaB45. Though this purifi-
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
tion (Figs. 2A and 3A); the lower band was never seen
in tissues extracted in hot SDS-PAGE buffer (Fig. 2, B
and C).
Characterization of VCaB45. Localization and
Distribution of VCaB45
Figure 2. A, Enrichment of VCaB45 during purification. Equal
amounts of each fraction (2 ␮g protein) were fractionated by SDSPAGE. A western blot was probed with a 1:2,000 (v/v) dilution of
antibody raised against purified VCaB45. Lane 1, Whole celery
(Apium graveolens) homogenate (VCaB45 is not detectable at this
exposure); lane 2, microsomal membranes; lane 3, vacuolar membrane fraction; lane 4, Triton X-100 extract of vacuole membrane
fraction (soluble vacuolar proteins); lane 5, peak fraction from DEAESepharose column. B, Various plant tissues contain proteins of similar molecular mass that are immunologically related to VCaB45.
Celery vacuolar membranes (0.05 ␮g, lane 1) and tissue homogenates of pea (Pisum sativum; lane 2), soybean (Glycine max; lane 3),
and maize (Zea mays; lane 4) seedlings (9 ␮g each), were probed
with anti-VCaB45. C, VCaB45 accumulates to high levels in cortical
tissues of celery petioles. Cortical (C) and vascular (V) tissues were
isolated separately and homogenized directly into 2⫻ SDS-PAGE
buffer. Gels were loaded with equivalent amounts of protein (10 ␮g)
and blots were probed with anti-VCaB45. Molecular mass standards
(in kD) are indicated on the right.
cation procedure was used for eliciting antisera, the
large-scale purification methods used in all subsequent experiments described in this paper are outlined
later. VCaB45 was susceptible to proteolytic degradaPlant Physiol. Vol. 130, 2002
Immunoreactive proteins were found in all dicotyledonous species tested (celery, pea, soybean, beets
[Beta vulgaris], potato [Solanum tuberosum], carrot
[Daucus carota], tobacco [Nicotiana tabacum], and Arabidopsis), but were markedly absent in the two
monocotyledonous species (maize and oats [Avena
sativa]) tested (Fig. 2B). To determine whether
VCaB45 protein levels might be correlated with the
presence of vacuoles, we exploited the characteristic
of celery petioles that allows, with relative ease, the
physical separation of vascular tissues from cortical
tissue. The parenchyma cells in the cortical tissues
tend to be extensively vacuolated, whereas the cells
in the vascular tissues are much less so. VCaB45 was
observed to be at low levels in vascular tissues but
was highly abundant in the cortical tissues, consistent with a vacuole location and function (Fig. 2C).
VCaB45 appears to be present predominantly in the
low-density fractions (Fig. 3, 0%/4% [w/w] dextran
interface) enriched in vacuole membranes (Randall,
1992). Although significant amounts of VCaB45 also
appear in higher density gradient fractions (4%/7%
[w/w] dextran), it is not clear whether this is due to
the presence of vacuole membranes in this fraction or
whether VCaB45 is also localized in the endoplasmic
reticulum (which is enriched in the 4%/7% [w/w]
fraction). The low-density location is consistent
with observations of the calcium-binding activity
(observed at 45 kD) made previously (Randall, 1992).
To further examine the subcellular localization of
VCaB45, we took an additional approach. We had
previously developed methods to evacuolate tobacco
protoplasts from suspension-cultured tobacco BY2
cells and to isolate intact vacuoles (Seals and Randall,
1997). Using these methods, vacuole markers in
evacuolated protoplasts were reduced 10- to 12-fold
and in isolated vacuoles were enriched approximately 3- to 4-fold (Seals and Randall, 1997). Consistent with a vacuole location, a tobacco protein (Tb45),
immunologically related to the celery VCaB45, is
largely depleted in evacuolated protoplasts and, conversely, is enriched in isolated vacuoles from tobacco
cells (Fig. 3B). However, based on all these data, we
cannot conclude that VCaB45 localization is restricted to the vacuole.
VCaB45 could be quantitatively removed from the
low-density membranes by treatment with 0.2%
(w/w) Triton X-100 (Fig. 3A), a concentration insufficient to solubilize integral membrane proteins (Randall and Sze, 1986). Treatment of membranes with 0.5
m KI, a chaotropic agent often used to dissociate
peripheral proteins from membranes (Lai et al.,
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
677
Heyen et al.
Figure 3. A, VCaB45 is enriched in low-density fractions and is released by 0.2% (w/w) Triton X-100. Membranes obtained
from dextran step gradients (0%/4% [w/v] dextran interface, 4%/7% [w/v] dextran interface, etc., see Randall, 1992) were
permeabilized with 0.2% (w/w) Triton X-100 (for 30 min at 4 C) and then centrifuged at 214,200g for 40 min. Equivalent
portions of untreated membranes (M), the Triton X-100-solubilized supernatants (S), and membrane pellets (P) were
separated by SDS-PAGE. The western blot was probed with anti-VCaB45. B, VCaB45-related protein (designated Tb45) is
depleted in evacuolated tobacco protoplasts and is enriched in isolated vacuoles. Protoplasts (P) were produced from
tobacco BY2 cells and vacuoles were selectively removed by ultracentrifugation resulting in evacuolated protoplasts (EV).
In a separate preparation, vacuoles (V) were isolated and purified from protoplasts (P). Identical quantities of protein were
resolved by SDS-PAGE. Tb45 indicates a tobacco protein immunologically related to celery VCaB45. Blots were probed with
anti-VCaB45. Molecular mass standards (in kD) are indicated on the right.
1988), did not release VCaB45 (data not shown).
These data are consistent with either a lumenal location or a very weak hydrophobic association of
VCaB45 with the membrane. To better understand
the potential functions of VCaB45, we determined
whether this protein was disposed on the cytosolic
side or the lumenal side of the vacuole membrane.
We first needed to determine that the low amount of
Triton X-100 could release lumenally localized proteins. Treatment of membranes with 0.2% (w/w) Triton X-100 released 8.5-fold more of the lumenal enzyme acid phosphatase and 6.4-fold more of the
VCaB45 protein than membranes not treated with
detergent (Fig. 4A). The small amount of activity
solubilized in the untreated membranes is likely due
to either membrane damage during the initial membrane preparation or the subsequent centrifugation
steps. Overall, the data are consistent with Triton
X-100-permeabilized membranes releasing lumenally
localized proteins. To further define the location of
VCaB45, freshly isolated membranes were treated
with proteinase K (Fig. 4B). If VCaB45 was located
inside the membrane vesicle, then one would expect
no protein degradation unless the membranes were
disrupted by detergent. In the detergent-treated
membranes, VCaB45 was readily proteolysed to a
smaller size by proteinase K (at higher concentrations
of protease VCaB45 was completely degraded; data
not shown). The purified VCaB45 was also susceptible to proteinase K digestion (data not shown). However, in the absence of detergent, the insensitivity of
VCaB45 to the protease suggests the membrane is
678
protecting it and, thus, argues strongly for a lumenal
location of VCaB45.
Sequence and General Properties of VCaB45
After a cyanogen bromide cleavage and subsequent
sequencing by in-line HPLC/mass spectrometry, we
obtained a mixed sequence, (M) EKEDEKLPGGVKTVE (major species) and (M) KKIKHDXSKVGAKTF
(minor species). These sequences were searched utilizing the program FASTF (searching the database National Center for Biotechnology Information/BLAST
NR). The FASTF algorithm takes various combinations of the two mixed sequences and searches the
database finding the best match. The top match obtained was found within a carrot sequence (carrot
dehydrin, accession no. AB010898) MEKIKEKLPGGGKKVE, a perfect match (Table I). Note that this
sequence contains a close match to the canonical “K”
domain, the dehydrin signature motif (EKKGIMDKIKEKLPG; Close et al., 1993a, 1993b; Close, 1997).
The carrot dehydrin does not contain any perfect “K”
domain (Tan et al., 2000). The remaining amino acids
matched a second sequence in the same protein (MKKEEKDETKVIATEF, 10 amino acids identical, four
similar, one not similar, and one amino acid unidentified in the experimentally obtained sequence). Overall, these two matches represent 26 identical amino
acids of a total of 32 amino acids (81% identity) and 30
similar amino acids of 32 total (94% similarity). In
addition, the top 10 sequences returned from the database search were dehydrins from various organisms.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
Figure 4. Localization of VCaB45 in vacuole membranes. A, Supernatants of vacuole membranes treated with or without 0.2% (w/w)
Triton X-100, were obtained after centrifugation at 100,000g for 30
min. Supernatants were separated by SDS-PAGE, blotted, and probed
with anti-VCaB45. Note that to visualize VCaB45 in untreated membrane supernatants it was necessary to load twice the proportion of
that loaded for Triton-treated supernatants. The numerical data for
VCaB45 represent densitometric analysis (arbitrary units) of the western blot, factoring the different gel loads. Acid phosphatase activity of
the supernatants of membranes treated either with or without Triton
X-100 were 17 and 2 A405 min⫺1 ␮l extract⫺1, respectively. B,
Vacuole membranes were treated with or without 0.2% (w/w) Triton
X-100 and simultaneously with or without 2 mg mL⫺1 proteinase K.
After a 30-min incubation at 4°C, a portion of the entire sample was
separated by SDS-PAGE, blotted, and probed with anti-VCaB45.
Molecular mass standards (mass in kD) are shown at the left.
Further, in the recent screening of an Arabidopsis
expression library with the antibody raised against
celery VCaB45 (S.K. Randall, unpublished data), we
have obtained only cDNA clones that encode a dehydrin protein (i.e. ERD14). Based on these data and
together with reactivity to the anti-K domain serum
(see below), we have concluded that celery VCaB45 is
a dehydrin-like protein.
One prediction of VCaB45 properties based upon
its hypothetical identity as a dehydrin-like protein is
that of solubility after heat treatment. A number of
the dehydrin family members remain soluble after a
90°C heat treatment (Lin et al., 1990; Close et al.,
1993b). VCaB45 remained soluble after heat treatment (Fig. 5A). We took advantage of the solubility
after heat treatment of VCaB45 to develop an alternative (rapid, with little proteolytic breakdown) purification procedure for VCaB45. This procedure involved isolation of vacuole-enriched membranes,
extraction with 0.2% (w/w) Triton X-100, heat treatPlant Physiol. Vol. 130, 2002
ment of the extract, recovery of the soluble phase after
heat treatment, and, finally, anion-exchange chromatography. A substantial enrichment of VCaB45 was
achieved through this heat treatment procedure (Fig.
5, A and B). In addition, the calcium-binding activity
(Fig. 5C) was conserved during this procedure and
was consistent with the enrichment of the immunoreactive polypeptide. The overall increase in purification efficiency facilitated the processing of the approximately 8 kg of petiole material (per experiment)
required for the calcium-binding studies discussed
below.
We do not have the full-length sequence for VCaB45
and because several dehydrins behave anomalously in
SDS-PAGE, we determined the mass of purified
VCaB45 by matrix-assisted laser-desorption ionization
time of flight (MALDI-TOF). The major molecular ion
was found to be 16.449 kD, whereas a minor species
(13% of the major) was 19.043 kD. No larger molecular
species were observed. We conclude it likely that the
true mass of VCaB45 is approximately 16.5 kD. This
overestimate of mass by SDS-PAGE is consistent with
that of other dehydrins (Gilmour et al., 1992; Welin et
al., 1995; Svensson et al., 2000).
The confirmation of VCaB45’s identity as a
dehydrin-like protein suggested protein levels might
be regulated by environmental factors. In seedlings,
VCaB45 levels are increased by cold stress, the phytohormone abscisic acid (ABA), and by drought
stress (Fig. 6). However, mature celery plants did not
regulate levels of VCaB45 by cold stress (Fig. 6). The
regulation by environmental stress in seedlings is
consistent with VCaB45’s proposed identity as a
dehydrin-like protein.
Phosphorylation of VCaB45 and
Calcium-Binding Properties
The dehydrin Rab17 can be phosphorylated in the
“S” domain (Vilardel et al., 1990; Plana et al., 1991;
Jensen et al., 1998) and most dehydrins have potential CKII phosphorylation sites (in the “S” domain).
Because unpublished data from our lab suggested
other dehydrins were also phosphorylated, we decided to test whether VCaB45 was phosphorylated
and, if so, whether the phosphorylation state of the
enzyme might influence calcium binding. Treatment
of celery VCaB45 with shrimp alkaline phosphatase
(SAP) resulted in a small but discernible shift in
apparent molecular mass (of approximately 4–6 kD)
on SDS-PAGE gels, whereas denatured SAP had no
effect (Fig. 7A). Such shifts are often used to assess
the phosphorylation status of proteins (Kitta et al.,
2001; Rivedal and Opsahl, 2001). The action of alkaline phosphatase on VCaB45 could be prevented by
200 ␮m vanadate, an inhibitor of phosphatase activity
(Fig. 7B, I), indicating the gel shift requires an enzymatically active phosphatase. These results suggest
that the purified VCaB45 is phosphorylated. A logical
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
679
Heyen et al.
Table I. A mixed amino acid sequence obtained from a cyanogen bromide digest and subsequent sequencing by in-line HPLC/mass spectrometry of VCaB45 is aligned with the carrot dehydrin sequence (accession no. AB010898)
The Mets were inferred (cyanogen bromide cleaves peptides after Mets). When these sequences were searched on GenBank (plant sequences,
nonredundant, utilizing the program FASTF) the greatest similarity was found with the carrot sequence, which encodes a dehydrin protein. Out
of the mixed sequence was obtained a perfect match (VCaB45 Seq 2, from amino acids 142–157) and a second match (VCaB45 Seq 1, from
amino acids 29 – 44), using the remaining amino acids, with 62% identity and 87% similarity. The alignment between the carrot dehydrin and
the two celery VCaB45 sequences are highlighted in black (identical amino acids) and in gray (similar amino acids).
extension of this conclusion is that this protein is
normally phosphorylated in planta. Even more exciting is the observation that treatment of VCaB45 with
alkaline phosphatase greatly reduces the ability of
this protein to bind calcium when measured by the
calcium ligand blot method (Fig. 7B, II). The presence
of potential casein kinase II phosphorylation sites on
other known dehydrins suggested to us that VCaB45
might also be phosphorylated by CKII. It appears
that VCaB45 is an “in vitro” substrate for CKII and
that the phosphorylation of previously dephosphorylated VCaB45 shifts the protein back to a higher
apparent molecular mass, and restores, at least in
part, the calcium-binding activity (Fig. 7C).
To confirm that the polypeptide that shifted after
treatment with alkaline phosphatase was the same
dehydrin-like protein, an antibody specific to dehydrins was utilized (Fig. 7D). The dehydrin antibody
detected a similar shift as that observed with the
VCaB45 antibody (Fig. 7D). The specificity of the
antibody interaction was confirmed by the reaction
against the known Arabidopsis dehydrin, ERD14,
and competition with the K peptide. The antibody
detection of both ERD14 and VCaB45 was prevented
by the blocking K peptide (Fig. 7D). It is also significant that the antibody raised against VCaB45 recognized ERD14, consistent with the identity of VCaB45
as a dehydrin-like protein. The reactivity of the antibody raised against the K peptide (the signature sequence of dehydrins) confirmed that the shifting phenomena was due to a change in the phosphorylation
status of the dehydrin-like protein, VCaB45, which
resulted in altered calcium binding (Fig. 7, B and C).
Thus, the calcium-binding activity of VCaB45 is influenced by its phosphorylation status.
680
The initial characterization of VCaB45 was based
on the premise that calcium binding measured by the
ligand-blot assay reflects a “true” functional activity.
This method has been used in the past to identify a
variety of calcium-binding proteins (Ostwald and
MacLennon, 1974; Campbell et al., 1983a, 1983b). We
have also compared calcium-binding activity (on ligand blots) with a vacuolar annexin protein, VCaB42
(Seals and Randall, 1994, 1997). Although the vacuolar annexin binds calcium in the native state, with an
apparent dissociation constant (Kd) of 60 nm (Seals
and Randall, 1994), it does not bind calcium by the
calcium ligand-blot assay (Fig. 8). This is consistent
with the known properties of the endonexin fold, the
calcium-binding domain found in the family of annexin proteins. This analysis also demonstrates that
VCaB45 binds calcium with some degree of specificity (also note the paucity of calcium-binding proteins
in crude preparations; Fig. 5). Although the blotbased ligand-binding assay has been very convenient
for the initial identification of calcium-binding proteins and for rapid analysis during purification, it
does not give information about the calcium-binding
activity of the native protein. To quantitatively analyze the calcium-binding parameters of native
VCaB45, we have conducted equilibrium dialysis
calcium-binding assays.
Equilibrium dialysis experiments indicated apparent saturable binding of calcium to native VCaB45
(Fig. 9A). Higher calcium concentrations than those
shown in Figure 9 encouraged precipitation of
VCaB45. Scatchard plots (Fig. 9D) indicated the presence of two distinct binding sites of different affinity.
Average values for the Kds of these sites were 0.22 ⫾
0.142 and 0.64 ⫾ 0.08 mm (average of nine experi-
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
Figure 5. Comparison of calcium-binding activity and total protein obtained after heat treatment. A, Celery VCaB45 remains soluble after
heat treatment. The Triton X-100 extract (Total)
was heat treated (20 min at 80°C–90°C) and
then chilled to 4°C (10 min). A supernatant and
a pellet were obtained after centrifugation of the
extract at 100,000g for 30 min. Equal portions of
all fractions were separated by SDS-PAGE and
blots probed with anti-VCaB45. Greater than
90% of total protein precipitated after the heat
treatment (not shown). B, Lane 1 contained 22.5
␮g of protein; the other lanes contained a corresponding portion equivalent to the membrane
volume containing 22.5 ␮g of protein. B, Onemillimeter-thick gel stained with Coomassie
Brilliant Blue. C, The same samples (but twice
the amounts of protein were loaded compared
with the gel in B) separated on a 2-mm-thick
SDS-PAGE gel and 45calcium ligand-blotted.
Lanes 1, 2, and 3 are equivalent fractions in both
B and C. Lane 1, 0%/4% (w/v) dextran membranes; lane 2, Triton-extracted supernatant;
lane 3, heat-treated supernatant. Though run on
different gels, the major Coomassie-staining
band in B, lane 3 corresponds to the calciumbinding band (arrow indicates VCaB45) in C,
lane 3 (by protein staining of the polyvinylidene
difluoride [PVDF] blot after the calcium blot;
data not shown). Molecular mass standards (B,
stainable; C, prestained; Bio-Rad Laboratories,
Hercules, CA) are indicated on the left. Arrowhead indicates VCaB45.
ments using three different preparations of purified
VCaB45). Estimates of the number of binding sites on
VCaB45 were 24.7 ⫾ 11.7 mol calcium bound per
mole VCaB45 (assuming the mass of VCaB45 is 16.5
kD). A Hill plot (Fig. 9B) indicated little cooperativity
between these multiple binding sites because the
slope was near 1.
The phosphorylation state of VCaB45 had a dramatic effect on the calcium-binding activity. Dephosphorylation of the purified VCaB45 resulted in a
large decrease (Fig. 9, A and C) in calcium binding
(corresponding to a 30–100-fold decrease in calcium
bound per mole VCaB45). Similar to the ligand blotting results, rephosphorylation of VCaB45 with casein kinase II resulted in a modest recovery of
calcium-binding activity (Fig. 9C). The phosphorylation state seems to similarly affect both the high- and
low-affinity (measured at 0.1 or 0.8 mm calcium)
calcium-binding sites.
Various ions were tested for the ability to compete
for VCaB45 calcium-binding sites (Fig. 10). Magnesium and the monovalent cations had little effect on
Plant Physiol. Vol. 130, 2002
calcium binding to VCaB45. However, a variety of
divalent cations (at 0.2 mm) did decrease calcium
binding. Zinc gave the greatest amount of inhibition
(of the divalent cations; Fig. 10 and inset). Zinc interaction with the calcium-binding site was not surprising because zinc seems to have affinity for a number of
calcium binding sites in a variety of proteins. Of those
divalent cations tested, only manganese and magnesium are likely to be physiological ligands (though
manganese is toxic at millimolar levels; Foy et al.,
1978). The concentration at which the other cations
were tested is unlikely to be achieved in living plants.
Lanthanum (a trivalent cation), an often-used nonphysiological inhibitor for calcium-binding sites and
in particular calcium channels (Friedman et al., 1998;
Dennison and Spalding, 2000), was strongly inhibitory
to calcium binding by VCaB45.
DISCUSSION
VCaB45 is likely a vacuole-localized member of the
dehydrin protein family. In support of that conclu-
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
681
Heyen et al.
Figure 6. Expression of VCaB45 protein in celery
during low temperature (LT), ABA, and drought
(DR) treatments. Older celery plants were treated
at 6°C for 1 week and younger seedlings at 5°C
for 2 d, 100 ␮M ABA (in 0.01% [v/v] Tween 20,
0.26% [v/v] methanol, sprayed on leaves 2 successive d) for 2 d, and drought stress (not watered
for 11 d). Age indicated is the time after planting
(germination is approximately 2 weeks after
planting). The minus signs indicate the respective
controls for each of the treatments. Total proteins
were extracted as described in “Materials and
Methods.” A, Blots were probed with antiVCaB45. Left, Older plants; performed in a different experiment and the blots were developed
separately from those shown in the younger
plants. Both of these experiments were performed
at least twice with similar results. B, Coomassiestained gel indicating total protein from the plants
treated in A. The major band present is Rubisco
large subunit, mass of approximately 53 kD.
sion is an array of evidence substantiating the similarities between VCaB45 and dehydrins: (a) VCaB45
contains a region that is very similar to the dehydrin
“signature” motif, the “K” domain, and another region very similar to a K-rich sequence in a carrot
dehydrin (Table I); (b) VCaB45 is specifically recognized by an antibody raised to the K domain (Fig.
6D), and, conversely, an antibody raised against
VCaB45 recognized a known dehydrin; (c) When an
Arabidopsis expression library was screened with
the monospecific antibody for VCaB45, only
dehydrin-encoding cDNAs were obtained (ERD14;
S.K. Randall, unpublished data); (d) VCaB45 is soluble after heating to denaturing conditions (90°C for
20 min), a characteristic of the dehydrin family; (e)
VCaB45 shows an anomalous migration on SDSPAGE, a characteristic of some dehydrins; and (f) The
accumulation of VCaB45 in response to the various
environmental stresses is also consistent with the
pattern of dehydrin expression. Both data from density gradients (in celery) and the isolation of intact
vacuoles (from tobacco) suggest that VCaB45 is at
least partially vacuole localized. The proteolysis protection experiments and the 0.2% (w/w) Triton X-100
extractability are consistent with a lumenal location
of this protein.
Although dehydrins were initially discovered over
a decade ago, the physiological function of dehydrins
has remained enigmatic. It has been speculated that
dehydrins might bind ions (particularly phosphate or
sulfate ions; Dure, 1993), or stabilize (chaperone) cytoplasmic proteins against denaturation, or stabilize
supermolecular structures or membranes (Close et
al., 1993a, 1993b; Kaye and Guy, 1995; Close, 1997).
We now have evidence that the celery dehydrin-like
682
protein binds calcium. An important question is
whether ion binding is a general property of dehydrins. This hypothesis appears to be consistent with
the purification of dehydrins on immobilized metal
columns (Svensson et al., 2000).
Although most members of the dehydrin superfamily of related genes are transcriptionally activated during periods of osmotic stress (Skriver and
Mundy, 1990; Close, 1993b, 1997), several dehydrins
appear to be constitutively expressed (Welin et al.,
1994; Nylander et al., 2001). It is interesting that
mature celery plants appear to constitutively express VCaB45, whereas in young seedlings, levels
of VCaB45 are responsive to environmental stress
(Fig. 7).
Calcium Binding as a Property of VCaB45
The VCaB45 protein has a high capacity for calcium
(binding an average of 25 mol calcium mol protein⫺1)
and has at least two distinct types of binding sites
with Kds of approximately 0.22 and 0.64 mm. Calcium
buffer proteins like calreticulin, calsequestrin, and
calnexin use extensive arrays of acidic amino acidrich regions (at the carboxyl terminus) to bind a large
number of calcium ions per protein molecule. Calreticulin has a single high-affinity (1.6 ␮M) calciumbinding site in the P domain (Pro rich) and highcapacity/low-affinity (0.3–2 mm) binding sites at the
C terminus (Michalak et al., 1992). The latter site is
characterized by 37/55 residues that are acidic. Consistent with the acidic nature of calcium-binding sites
in calreticulin, VCaB45 is a rather acidic protein,
having a pI of 5.2. Although manganese and zinc are
likely candidates to bind VCaB45 based on in vitro
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
Figure 7. Treatment of purified celery VCaB45 with SAP results in a shift in apparent molecular mass and a decrease in
calcium-binding activity. A, VCaB45, purified by heat treatment followed by anion-exchange chromatography, was treated
for 60 min with SAP. Controls (0 time) were obtained by adding SAP and immediately adding SDS-PAGE sample buffer and
boiling. After SDS-PAGE, blots were probed with anti-VCaB45. B, VCaB45 was treated for 60 min as in A, either without
SAP, with SAP, or with SAP plus 200 ␮M sodium (ortho) vanadate. Reactions were terminated by the addition of hot
SDS-PAGE sample buffer and heating at 90°C for 5 min. After SDS-PAGE, gels were stained with Coomassie Brilliant Blue
(I) or used for calcium ligand blots (II). C, VCaB45 was dephosphorylated with SAP as in A and B, then was repurified by
anion-exchange chromatography, and incubated for 3 h at 30°C in the presence or absence of casein kinase II (CKII).
Samples were analyzed by Coomassie staining of gels (I) or by calcium ligand blots (II). D, Gel shifts monitored by
anti-VCaB45 and antidehydrin (DHN). The first two lanes of each panel contained VCaB45, whereas the third contained the
purified Arabidopsis dehydrin ERD14. VCaB45 was treated with SAP as in B. Competition of the anti-DHN was with 2.5 mg
mL⫺1 K peptide (TGEKKGIMDKIKEKLPGQH). ERD14 (Arabidopsis ecotype Columbia) was expressed in Escherichia coli and
purified by heat treatment followed by anion-exchange chromatography.
assays (Fig. 10), both of these are micronutrients and
are unlikely to reach millimolar concentrations in
plant cells. Although magnesium can be found in
cells at millimolar levels, it does not appear to compete as well for calcium binding (Fig. 10). Thus, the
predominant physiologically relevant ligand for
VCaB45 appears to be calcium. For these reasons, we
suggest that the physiological role of VCaB45 may
involve calcium binding. Because VCaB45 is a
dehydrin-like protein, it is of great interest to determine whether other proteins of the dehydrin family
also bind calcium and whether calcium binding is a
distinct property related to the function of dehydrins.
We are presently testing that hypothesis.
Plant Physiol. Vol. 130, 2002
Regulation of Calcium Binding by Phosphorylation
Because calcium-binding properties of VCaB45 are
dependent on its phosphorylated state, we were interested in whether the phosphorylation of VCaB45
was regulated by stress conditions. We addressed
this question indirectly by examining the apparent
mass of VCaB45 under cold stress conditions or when
it is constitutively expressed (and additionally exposed to cold stress). We could visualize the phosphorylation status by a shift in apparent mass (Fig.
7). By this method, under all conditions we have
tested, VCaB45 is always present in vivo in the phosphorylated state (Fig. 6). We see no evidence of a
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
683
Heyen et al.
MATERIALS AND METHODS
Plant Material and Vacuole Isolation
Figure 8. VCaB45 binds calcium in the ligand blot but the vacuole
annexin, VCaB42, does not. VCaB42 was obtained as previously
described (Seals and Randall, 1994) and loaded in the first lane of
each gel. VCaB45 was eluted from a nitrocellulose spot cut from a
two-dimensional gel. The nitrocellulose was boiled in SDS-PAGE
buffer and loaded directly into the well with the sample buffer (the
second lane of each gel). Left, Coomassie-stained SDS-PAGE. Right,
Calcium ligand blot. Position of molecular mass standards (in kD) are
indicated.
cold-induced alteration of dephosphorylation or
phosphorylation. However, it is important to note
that subtle changes in phosphorylation would be
difficult to detect using this assay. At this time, it is
not clear whether the phosphorylation of VCaB45 is a
reversible phosphorylation or whether it is a constitutive phosphorylation. The issue of where VCaB45
is phosphorylated, and by what kinase, are intriguing questions yet to be answered. Multiple perfect
casein kinase II phosphorylation sites {[ST]-x(2)[DE]} can be found in some dehydrins (four of eight
possible sites within or adjacent to the S domain in
ERD14, for example). The presence of these sites in
other dehydrins was our rationale for testing the
affect of CKII phosphorylation on calcium-binding
activity in VCaB45. Although CKII (a ubiquitous
kinase) could phosphorylate VCaB45 “in vitro” and
activate the protein to bind calcium, the reactivation
was incomplete (Fig. 9C). The reason for this incomplete activation is unclear. Regardless, the identity
of the activating phosphorylation target site may
help to illuminate the identity of the responsible
kinase.
Based upon our findings that VCaB45 can bind
calcium (in preliminary experiments, calcium binding has also been observed in other known dehydrins
(S.K. Randall, unpublished data), we propose two
alternative hypotheses for the function of VCaB45
specifically, and possibly dehydrins in general. First,
a protective function of these proteins may be conferred by their high capacity for calcium binding.
Thus, they may help alleviate the elevated intracellular calcium concentrations caused by leakage
across membranes that occurs during environmental
stress. Alternatively, they may play a protein chaperone role that is calcium dependent, similar to the
function of calnexin and calreticulin (Nigam et al.,
1994; Peterson et al., 1995; Hebert et al., 1996).
684
For most experiments, celery (Apium graveolens) was obtained from a
local grocery; for seedling experiments (Fig. 7), celery cv Tall Utah was used.
The procedure for vacuolar membrane isolation was as described in Randall
(1992) with slight modifications. Homogenization buffer composed of 250
mm d-mannitol, 3 mm EGTA, 50 mm HEPES, and 1 mm dithiothreitol (DTT),
pH 7.4. To reduce protease activities, a cocktail comprising 1 mm phenylmethylsulfonyl fluoride (0.05% [w/v] dimethylsulfoxide, final concentration), 1 mm benzamidine, 10 ␮g mL⫺1 aprotinin, 1 ␮g mL⫺1 leupeptin, and
10 ␮m pepstatin, was present during homogenization. Celery petioles were
homogenized in a blender (Waring, Winsted, CT) for 15 s at top speed with
a ratio of 2.5 mL of homogenization buffer per gram fresh weight petioles.
The supernatant obtained from two low-speed centrifugations (10,000g for
10 min) was centrifuged for 35 min at 58,000g. The membranes obtained in
the pellet were resuspended in 2.5 mm HEPES (pH 7.2), 250 mm d-mannitol,
and 1 mm DTT (resuspension buffer), and separated on dextran gradients.
The 0%/4% (w/w) dextran interface is highly enriched in vacuole membranes, the 4%/7% and 7%/12% (w/w) dextran interfaces are enriched in
the endoplasmic reticulum, and the membranes pelleted thorough the 12%
(w/v) dextran cushion are enriched in chloroplasts and mitochondria (Randall, 1992). Dextran gradient fractions were diluted and recentrifuged. The
pellets were resuspended in resuspension buffer and stored at ⫺80°C. For
purposes of producing homogenates with minimal proteolysis, tissues were
homogenized directly into hot 2⫻ SDS-PAGE sample buffer, at 1 mL g fresh
weight⫺1 plant material. The homogenates were centrifuged for 10 min at
10,000g. Supernatants obtained were boiled for 5 min, and then stored at
⫺80°C. In this case, estimates of protein were made using the Amido Black
assay (Kaplan and Pedersen, 1985).
Triton X-100 Permeabilization of the
Vacuole Membranes
The highly enriched vacuolar membrane fraction (see Randall, 1992) was
permeabilized with a low concentration of Triton X-100 detergent to release
the contents of the vacuole membrane vesicles. The vacuolar membrane
fraction was treated with 0.2% (w/w) Triton X-100 and 1 mm DTT for 30 min
at 4°C, with gentle rotation. Permeabilization was conducted in the presence
of the proteinase inhibitor cocktail described above. On some occasions, the
Triton X-100-treated membranes were sonicated for 4 min and then centrifuged for 30 min at 214,200g at maximal radius (type 90 Ti rotor). Later, it
became clear that sonication was unnecessary. The resulting supernatant,
containing soluble lumenal vacuolar proteins, was saved for further purification steps and stored at ⫺80°C.
For the localization experiments, fresh vacuolar membranes isolated from
the 0%/5% (w/w) dextran gradient were treated (or not) with 0.2% (w/w)
Triton X-100 and then centrifuged at 135,000g (at maximal radius, in a TL
Ultracentrifuge, Beckman Instruments, Fullerton, CA) for 1 h at 4°C. The
supernatants were recovered and diluted 100-fold for the acid phosphatase
assay or analyzed by SDS-PAGE. In some cases, membranes were treated
with proteinase K in the presence or absence of 0.2% (w/w) Triton X-100.
The digestion was stopped by the addition of phenylmethylsulfonyl fluoride (5 mm final concentration) and then the samples were adjusted to 1⫻
SDS-PAGE sample buffer and boiled.
Fractionation of Triton X-100 Supernatant
(Lumenal Vacuolar Contents) by
Anion-Exchange Chromatography
The Triton X-100 supernatant was diluted 10-fold with 20 mm Tris-HCl
buffer, pH 8.2 (at 4 C), and loaded at 0.2 mL min⫺1 onto a 50-mL packed bed
volume of DEAE-Sepharose (Amersham-Pharmacia Biotech, Uppsala)
anion-exchange column. The proteins were eluted with a linear 0 to 500 mm
NaCl gradient generated by a Waters 650E Advanced Protein Purification
System (Millipore, Bedford, MA). All fractions were assayed for calciumbinding activity with the 45Ca⫹2 ligand-blot assay described previously
(Randall, 1992).
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
Figure 9. Calcium binding to purified VCaB45 estimated by equilibrium dialysis. A, Calcium binding was estimated by
equilibrium dialysis. Calcium binding to purified VCaB45 (white squares) is the average of three independent experiments
from a single preparation of purified VCaB45. The SAP-treated calcium-binding data (white circles) were obtained from two
experiments using the same preparation. Error bars represent SDs; where not shown, the SDs were smaller than the symbol
indicating the data point. B, Hill plot (log % VCaB45 bound/100% ⫺ % VCaB45 bound, plotted against log [Ca])k derived
from the data in A. Line drawn through the 50% bound region had a slope of 0.961, indicating little cooperativity in calcium
binding. C, VCaB45 was untreated (gray bar), treated with SAP to dephosphorylate the protein (black bars), or was
dephosphorylated followed by rephosphorylation with casein kinase II (hatched bar). Calcium binding was performed at
concentrations estimated to measure high-affinity sites (100 ␮M) or low-affinity sites (800 ␮M). D, Scatchard plot (nY/X versus
nY, where nY is mol calcium bound per mol VCaB45 and X is free calcium) derived from the data shown in A. Kds derived
from the shown Scatchard plot were 0.41 and 0.75 mM calcium, while the maximum number of binding sites was 27
(assuming mass of VCaB45 is 16.5 kD). Data from three different preparations of purified VCaB45 (nine experiments)
indicated average Kds of 0.22 ⫾ 0.142, 0.64 ⫾ 0.08, and the maximal number of binding sites was 24.7 ⫾ 11.7.
Molecular Mass Estimates
Mass estimates were performed on a Voyager-DE Pro MALDI-TOF (PerSeptive/Applied Biosystems, Foster City, CA) operated in a positive ion
mode with sinapinnic acid as the matrix.
Immunization Schedule and Western Blotting
The calcium-binding protein (VCaB45) purified by anion-exchange chromatography and subsequently on large-scale two-dimensional gels
(O’Farrell, 1975) was then transferred to nitrocellulose. Spots amounting to
Plant Physiol. Vol. 130, 2002
5 ␮g of protein were excised and dissolved in 100 ␮L of dimethyl sulfoxide.
The solubilized nitrocellulose/protein mixture was mixed well with 100 ␮L
of Freund’s adjuvant. Injections of approximately 1 ␮g of protein were in
Balb/C mice, intraperitoneally. Subsequent boosts were carried out every 3
to 4 weeks. Blood was drawn 10 to 14 d after each boost and was incubated
at 4°C overnight. Serum samples were collected after centrifugation of the
incubated blood for 5 min at 3,000g. Antidehydrin (DHN) antiserum and
dehydrin K peptideTGEKKGIMDKIKEKLPGQH were kindly supplied by Dr.
Timothy J. Close (Close et al., 1993a). For western blotting, samples were
separated by SDS-PAGE (10% [w/w] acrylamide), transferred to either
nitrocellulose or PVDF membranes and blocked with 5% (w/v) nonfat dry
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
685
Heyen et al.
for 5 min at room temperature. The reaction was initiated by the addition of
30 ␮L of 10 mm p-nitrophenyl phosphate. The reaction was stopped (after a
30-min incubation, previously determined to be within the linear range) by
the addition of 300 ␮L of 1.0 m Na2CO3. The absorbance was read at 405 nm.
Data was expressed as change in A min⫺1 ␮l extract ⫺1.
Alkaline Phosphatase and Kinase Treatments
Typically, purified VCaB45 (approximately 0.6 ␮g) was treated for up to
60 min with 2.4 units of SAP (Roche Diagnostics, Indianapolis) in a total
volume of 18 ␮L. The incubation buffer was composed of 0.05 m Tris-Cl and
5 mm magnesium chloride, pH 8.0, at 20°C and approximately 50 mm NaCl
contributed by the purified VCaB45. Proteinase inhibitors (final concentrations: 10 mm benzamidine, 5 ␮g mL⫺1 aprotinin, and 50 ␮g mL⫺1 leupeptin)
were also present. Denaturation of SAP was accomplished by heating for 15
min at 65°C. When VCaB45 was to be subsequently rephosphorylated by
CKII, the dephosphorylation mixture (scaled up to 75 ␮L) was first mixed
with anion-exchange beads (approximately 10 ␮L of packed volume of
DEAE-Sepharose) for 2 h at 4°C. Under these conditions, SAP bound to the
beads, whereas the majority of VCaB45 did not. The unbound fraction was
then heat treated (as above) to denature any residual SAP and cooled on ice.
To 22 ␮L of the heat-treated mixture, 3 ␮L of 10⫻ kinase buffer (supplied by
the manufacturer), proteinase inhibitors (as above), and 250 units of casein
kinase II (CKII, New England Biolabs, Beverly, MA) were added (final
volume 30 ␮L). The kinase reaction was initiated by the addition of ATP to
a final concentration of 5 mm and incubation was continued for 3 h at 30°C.
Figure 10. Inhibition of calcium binding by cations. Calcium binding was estimated by equilibrium dialysis. Calcium binding was
performed as described, in the presence of 0.2 mM calcium. Competition was examined by adding an additional 0.2 mM of the indicated metal ion. The valence of the metal added is indicated. The bar
value for an additional 0.2 mM calcium added was calculated based
upon a 2-fold dilution of isotope and the binding data from Figure 9.
Inset, Competition of calcium-binding by zinc. SDs (where greater
than 5%) are indicated. Data are average of three experiments.
milk in either Tris-buffered saline or phosphate-buffered saline. To demonstrate the competition by the K peptide (Fig. 6D), equal volumes of antiDHN serum and K peptide (5 mg mL⫺1) were mixed and pre-incubated at
room temperature for 30 min. Anti-mouse or anti-rabbit IgG conjugated to
horseradish peroxidase were used as secondary antibodies for anti-VCaB45
and anti-DHN, respectively. Antibody detection procedures were essentially as described by the manufacturers of Western Blot Chemiluminescence (PerkinElmer Life Sciences Inc., Boston).
Calcium-Binding Assays
Calcium-binding activity was determined using either a ligand-blotting
method or by equilibrium dialysis. Ligand blotting was performed after
SDS-PAGE separation and western-blot transfer to PVDF membranes as
described (Maruyama and Nonomura, 1984) with some modifications (Randall, 1992). Equilibrium dialysis assays for calcium binding were performed
with Quizsep Micro Dialyzer cells (all teflon surfaces, 100-␮L capacity,
Mid-West Scientific, St. Louis) and Spectra/Por molecular weight cutoff
(6,000–8,000) membranes. Typically, 50 to 75 ␮L (approximately 15 ␮g of
protein) of purified VCaB45 was dialyzed for 5 h against 20 mL of 20 mm
Tris-Cl (pH 7.0) at 4°C. To reduce the possibility of spurious ions originating
from glassware, polypropylene-ware, teflon dialysis cells, and highly purified water were utilized for these experiments. Generally added to the
dialysis buffer were 0.05 to 1.0 mm calcium chloride and 2 to 4 ␮Ci 45calcium
chloride. After dialysis, at least two 20-␮L aliquots were taken from the both
the dialysate and the dialysis buffer and counted by liquid scintillation.
Acid Phosphatase Assays
Acid phosphatase activity was determined as described previously
(Boller and Kende, 1979). In brief, 100 ␮L of membrane extract, 320 ␮L of
water, and 50 ␮L of 1 m succinic acid (pH 5.0 with NaOH) were incubated
686
ACKNOWLEDGMENTS
The molecular mass estimate by MALDI-TOF was kindly performed
courtesy of Dr. Jeffrey Patrick at (Eli Lilly and Company, Indianapolis). We
also thank Dr. Alan Mahrenhotz of the Biochemistry Biotechnology Facility
(Indiana University School of Medicine, Indianapolis) for the amino acid
sequencing and MALDI-TOF analysis. The antidehydrin and K peptide
were graciously supplied by Dr. Timothy Close (University of California,
Riverside).
Received January 12, 2002; returned for revision February 28, 2002; accepted
June 11, 2002.
LITERATURE CITED
Allen GJ, Sanders D (1995) Calcineurin, a type 2B protein phosphatase,
modulates the calcium-permeable slow vacuolar ion channel of stomatal
guard cells. Plant Cell 7: 1473–1483
Boller T, Kende H (1979) Hydrolytic enzymes in the central vacuole of plant
cells. Plant Physiol 63: 1123–1132
Camacho P, Lechleiter JD (1995) Calreticulin inhibits repetitive intracellular
calcium waves. Cell 82: 765–771
Campbell KP, MacLennan DH, Jorgensen AO (1983a) Staining of calciumbinding proteins, calsequestrin, calmodulin, troponin C, and S-100, with
the cationic carbocyanine dye, “stains-all.” J Biol Chem 258: 11267–11273
Campbell KP, MacLennan DH, Jorgensen AO (1983b) Purification and
characterization of calsequestrin from canine cardiac sarcoplasmic reticulum and identification of the 53,000 dalton glycoprotein. J Biol Chem
258: 1197–1204
Chen F, Hayes PM, Mulrooney DM, Pan A (1994) Identification and
characterization of cDNA clones encoding plant calreticulin in barley.
Plant Cell 6: 835–843
Close TJ (1997) Dehydrins: a commonality in the response of plants to
dehydration and low temperature. Physiol Plant 100: 291–296
Close TJ, Fenton RD, Moonan F (1993a) A view of plant dehydrins using
antibodies specific to the carboxyl terminal peptide. Plant Mol Biol 23:
279–286
Close TJ, Fenton RD, Yang A, Asghar R, DeMason DA, Crone DE, Meyer
NC, Moonan F (1993b) Plant responses to cellular dehydration during
environmental stress. Curr Top Plant Physiol 10: 104–118
Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St-Arnaud R,
Dedhar S (1997) Calreticulin is essential for integrin-mediated calcium
signalling and cell adhesion. Nature 386: 843–847
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 130, 2002
Vacuolar Calcium-Binding Protein, VCaB45
Dennison KL, Spalding EP (2000) Glutamate-gated calcium fluxes in Arabidopsis. Plant Physiol 124: 1511–1514
Dure L III (1993) Structural motifs in Lea proteins. Plant responses to
cellular dehydration during environmental stress. Curr Top Plant Physiol
10: 91–103
Foy CD, Chaney RL, White MC (1978) The physiology of metal toxicity in
plants. Annu Rev Plant Physiol 29: 511–566
Franceschi VR, Li X, Zhang D, Okita TW (1993) Calsequestrin-like calciumbinding protein is expressed in calcium-accumulating cells of Pistis stratiotes. Proc Natl Acad Sci USA 90: 6986–6990
Friedman H, Meir S, Rosenberger I, Halevy AH, Kaufman PB, PhilosophHadas S (1998) Inhibition of the gravitropic response of snapdragon
spikes by the calcium-channel blocker lanthanum chloride. Plant Physiol
118: 483–492
Gilmour SJ, Artus NN, Thomashow MF (1992) cDNA sequence analysis
and expression of two cold-regulated genes of Arabidopsis thaliana. Plant
Mol Biol 18: 13–21
Hebert DN, Foellmer B, Helenius A (1996) Calnexin and calreticulin promote folding, delay oligomerization, and suppress degradation of influenza hemagglutinin in microsomes. EMBO J 15: 2961–2968
Hepler PK, Wayne RO (1985) Calcium and plant development. Annu Rev
Plant Physiol 36: 397–439
Jensen AB, Goday A, Figueras M, Jessop AC, Page SM (1998) Phosphorylation mediates the nuclear targeting of maize Rab17 protein. Plant J 13:
691–697
Kaplan RS, Pedersen PL (1985) Determination of microgram quantities of
protein in the presence of milligram levels of lipid with amido black 10B.
Anal Biochem 150: 97–104
Kaye C, Guy CL (1995) Perspectives of plant cold tolerance: physiology and
molecular responses. Sci Prog 78: 271–299
Kitta K, Clement SA, Remeika J, Blumberg JB, Suzuki YJ (2001)
Endothelin-1 induces phosphorylation of GATA-4 transcription factor in
the HL-1 atrial-muscle cell line. Biochem J 359: 375–380
Krause KH, Chou M, Thomas MA, Sjolund RD, Campbell KP (1989) Plant
cells contain calsequestrin. J Biol Chem 264: 4269–4272
Lai S, Randall SK, Sze H (1988) Peripheral and integral subunits of the
tonoplast H⫹-ATPase from oat roots. J Biol Chem 263: 16731–16737
Li X, Su RTC, Hsu H-T, Sze H (1998) The molecular chaperone calnexin
associates with the vacuolar H⫹-ATPase from oat seedlings. Plant Cell 10:
119–130
Lin CT, Gou WW, Everson E, Thomashow MF (1990) Cold acclimation in
Arabidopsis and wheat: response associated with expression of related
genes encoding “boiling stable” polypeptides. Plant Physiol 94:
1078–1083
Lu G, Sehnke PC, Ferl RJ (1994) Phosphorylation and calcium binding
properties of Arabidopsis GF14 brain protein homolog. Plant Cell 6:
501–510
Machlon AES (1984) Calcium fluxes at plasmalemma and tonoplast. Plant
Cell Environ 7: 423–429
Maruyama K, Nonomura Y (1984) High molecular weight calcium-binding
protein in the microsome of scallop striated muscle. J Biochem 96:
859–870
Mery L, Mesaeli N, Michalak M, Opas M, Lew DP, Krause KH (1996)
Overexpression of calreticulin increases intracellular calcium storage and
decreases store-operated calcium influx. J Biol Chem 271: 9332–9339
Michalak M, Miller RE, Burns K, Opas M (1992) Calreticulin. Biochem J
285: 681–692
Miller AJ, Vogg G, Sanders D (1990) Cytosolic calcium homeostasis in
fungi: roles of plasma membrane transport and intracellular sequestration of calcium. Proc Natl Acad Sci USA 87: 9348–9352
Napier RM, Trueman S, Henderson J, Boyce JM, Hawes C, Fricker MD,
Venis MA (1995) Purification, sequencing, and functions of calreticulin
from maize. J Exp Bot 46: 1603–1623
Plant Physiol. Vol. 130, 2002
Nelson DE, Glaunsinger B, Bohnert HJ (1997) Abundant accumulation of
the calcium-binding molecular chaperone calreticulin in specific floral
tissues of Arabidopsis thaliana. Plant Physiol 114: 29–37
Nigam SK, Goldberg AL, Ho S, Rohde MF, Bush KT, Sherman MYU
(1994) A set of endoplasmic reticulum proteins possessing properties of
molecular chaperones includes calcium-binding proteins and members of
the thioredoxin superfamily. J Biol Chem 269: 1744–1749
Nylander M, Svensson J, Palva ET, Welin BV (2001) Stress-induced accumulation and tissue specific localization of dehydrins in Arabidopsis.
Plant Mol Biol 45: 263–279
O’Farrell PH (1975) High resolution two-dimensional electrophoresis of
proteins. J Biol Chem 250: 4007–4021
Ostwald TJ, MacLennon DH (1974) Isolation of a high affinity calciumbinding protein from sarcoplasmic reticulum. J Biol Chem 249: 974–979
Peterson JR, Ora A, Van PN, Helenius A (1995) Transient, lectin-like
association of calreticulin with folding intermediates of cellular and viral
glycoproteins. Mol Biol Cell 6: 1173–1184
Plana M, Itarte E, Eritja R, Goday A, Pages M, Martinez MC (1991)
Phosphorylation of the maize RAB-17 protein by casein kinase 2. J Biol
Chem 266: 22510–22514
Randall SK (1992) Characterization of vacuolar calcium-binding proteins.
Plant Physiol 100: 859–867
Randall SK, Sze H (1986) Properties of the partially purified tonoplast
H⫹-pumping ATPase from oat roots. J Biol Chem 261: 1364–1371
Rivedal E, Opsahl H (2001) Role of PKC and MAP kinase in EGF- and
TPA-induced connexin43 phosphorylation and inhibition of gap junction
intercellular communication in rat liver epithelial cells. Carcinogenesis
22: 1543–1550
Sanders D, Brownlee C, Harper JF (1999) Communicating with calcium.
Plant Cell 11: 691–706
Seals DF, Randall SK (1994) A 43 kDa annexin-like protein is associated
with plant vacuoles. Plant Physiol 106: 1403–1412
Seals DF, Randall SK (1997) A vacuole-associated annexin protein, VCaB42,
correlates with the expansion of tobacco cells. Plant Physiol 115: 753–761
Skriver K, Mundy J (1990) Gene expression in response to abscisic acid and
osmotic stress. Plant Cell 2: 503–512
Svensson J, Palva ET, Welin B (2000) Purification of recombinant Arabidopsis dehydrins by metal ion affinity chromatography. Protein Expr
Purif 20: 169–178
Sze H, Liang F, Hwang I, Cunan AC, Harper JF (2000) Diversity and
regulation of plant calcium pumps: insights from expression in yeast.
Annu Rev Plant Physiol Plant Mol Biol 51: 433–462
Tan S-K, Sage-Ono K, Kamada H (2000) Cloning and characterization of
ECPP44, a cDNA encoding a 44-kilodalton phosphoprotein relating to
somatic embryogenesis in carrot. Plant Biotechnol 17: 61–68
Vilardel J, Goday A, Freire MA, Torrent M, Martinez MC, Torre JM, Pages
M (1990) Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize. Plant Mol Biol 14: 423–432
Welin BV, Olson A, Nylander M, Palva ET (1994) Characterization
and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 26:
131–144
Welin BV, Olson A, Palva ET (1995) Structure and organization of two
closely related low-temperature-induced dhn/lea/rab-like genes in Arabidopsis thaliana L. Heynh. Plant Mol Biol 29: 391–395
Xing T, Williams LE, Nelson SJ, East JM, Hall JL (1994) Immunological
detection and localization of a calsequestrin-like protein in red beet and
cucumber cells. Protoplasma 179: 158–165
Yuasa K, Maeshima M (2000) Purification, properties, and molecular cloning of a novel calcium-binding protein in radish vacuoles. Plant Physiol
124: 1069–1078
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
687