Plant Physiol. (1998) 118: 817-825
A High-Affinity Ca2+ Pump, ECA1, from the
Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but
Not by
Thapsigargin1
Feng Liang2 and
Heven Sze*
Department of Cell Biology and Molecular Genetics, H.J. Patterson
Hall, University of Maryland, College Park, Maryland 20742
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ABSTRACT |
To
identify and characterize individual Ca2+ pumps, we have
expressed an Arabidopsis ECA1 gene encoding an
endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast
(Saccharomyces cerevisiae) mutant K616. The mutant
(pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on
Ca2+-depleted medium. Membranes isolated from the mutant
showed high H+/Ca2+-antiport but no
Ca2+-pump activity. Expression of ECA1 in endomembranes
increased mutant growth by 10- to 20-fold in Ca2+-depleted
medium. 45Ca2+ pumping into vesicles from
ECA1 transformants was detected after the
H+/Ca2+-antiport activity was eliminated with
bafilomycin A1 and gramicidin D. The pump had a high
affinity for Ca2+ (Km = 30 nM) and displayed two affinities for ATP
(Km of 20 and 235 µM).
Cyclopiazonic acid, a specific blocker of animal
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited
Ca2+ transport (50% inhibition dose = 3 nmol/mg
protein), but thapsigargin (3 µM) did not. Transport was
insensitive to calmodulin. These results suggest that this endoplasmic
reticulum-type Ca2+-ATPase could support cell growth in
plants as in yeast by maintaining submicromolar levels of cytosolic
Ca2+ and replenishing Ca2+ in endomembrane
compartments. This study demonstrates that the yeast K616 mutant
provides a powerful expression system to study the structure/function
relationships of Ca2+ pumps from eukaryotes.
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INTRODUCTION |
The role of Ca2+ in signaling and development is well
recognized in eukaryotes (Bush, 1995
; Clapham, 1995); however, the
regulation of cytosolic and organellar Ca2+ in plants is
still poorly understood. In plant cells the cytosol usually contains 30 to 600 nM free Ca2+ (Reed et al., 1992),
whereas the cell wall and intracellular stores, such as the ER and
vacuole, contain 1,000- to 10,000-fold higher concentrations. The
Ca2+ gradients across the PM and the intracellular
membranes are energized by primary Ca2+-ATPases and by
H+-coupled Ca2+ antiport (Hirschi et al.,
1996). A variety of signals induces transient increases in cytosolic
Ca2+ because of the opening of specific Ca2+
channels (Bush, 1995). Recent studies with Ca2+-indicator
dyes demonstrated that the perception of a nodulation signal results in
Ca2+ waves or oscillations in root-hair cells of alfalfa
(Ehrhardt et al., 1996). In frog oocytes inositol
1,4,5-trisphosphate induced repetitive Ca2+ waves; however,
the frequency of the waves increased in cells overexpressing a SER
Ca2+ pump (Camacho and Lechleiter, 1993). Because the
SERCA pumps cytosolic Ca2+ into endomembrane compartments,
intracellular Ca2+ pumps could be an important factor in
controlling Ca2+ oscillations in plant and in animal cells.
Ca2+ is also essential for tip growth of pollen tubes. A
recent study showed that the tip-focused intracellular
[Ca2+] gradient oscillates with the same period as growth
(Holdaway-Clarke et al., 1997). High levels of Ca2+ in
intracellular compartments serve a variety of essential functions. This
divalent cation is a cofactor for specific enzymes (Bush et al., 1989)
and can bind to several chaperones in the ER (Bergeron et al., 1994).
Thus, endoluminal Ca2+ supplied by Ca2+ pumps
on the ER, Golgi, and secretory vesicles could affect processing and
sorting and determine the ultimate fate of membrane and secreted proteins (Rudolph et al., 1989). Furthermore, nuclear cisternal Ca2+ controls nuclear pore permeability and thus regulates
transport across the nuclear envelope (Perez-Terzic et al., 1997).
Although high-affinity, Ca2+-pumping ATPases have been
identified in a variety of membranes, including the PM, ER, and
tonoplast, the characterization of individual pumps separate from other
related pumps has been difficult in many plant studies. Extensive
biochemical studies demonstrated that plants have two major types of
Ca2+ pumps with distinct properties. The PM-type
Ca2+-ATPase is stimulated by calmodulin (Bonza et al.,
1998), whereas the ER type is not (Bush, 1995; Hwang et al., 1997).
However, unlike the PM-bound Ca2+ pumps from animals, the
PM-type Ca2+-ATPase is localized to several membranes in
plants (Askerlund, 1997; Hwang et al., 1997), suggesting a family of
calmodulin-stimulated pumps. Several genes encoding
Ca2+-ATPase homologs from plants have been isolated. Based
on similarities of the deduced amino acid sequence with animal
Ca2+ pumps, LCA1 from tomato (Wimmers et al., 1992)
and a gene from rice (Chen et al., 1997) encoded ER-type
Ca2+ pumps. PEA1 from Arabidopsis (Huang et al.,
1993) and BCA1 from cauliflower (Malmstrom et al., 1997)
encoded a PM-type Ca2+-ATPase homolog. Except for BCA1, the
biochemical activities of these gene products have not been
demonstrated.
To study individual Ca2+ pumps, we recently identified two
distinct Ca2+-ATPases by functional expression of two plant
genes in a yeast triple mutant. One cDNA (ECA1) encoded
an ER Ca2+-ATPase
homolog from Arabidopsis (Liang et al., 1997), and another gene
(ACA2, accession no. AF025842) encoded a PM-type
Ca2+-ATPase with a unique N-terminal regulatory domain
(Harper et al., 1998). ECA1 encoded a 116-kD
polypeptide that was more homologous to animal SERCA than to PM-type
Ca2+ pumps. When ECA1 was expressed in a
yeast triple mutant defective in both a Golgi and a vacuolar
Ca2+ pump (pmr1pmc1cnb1) (Cunningham and
Fink, 1994), growth of this mutant in Ca2+-depleted medium
was restored. Furthermore, ECA1 could be phosphorylated in a
Ca2+-dependent manner in vitro similarly to phosphoenzyme
intermediates formed in the reaction cycle of Ca2+-ATPases.
Therefore, ECA1 encoded a Ca2+-dependent
ATPase (Liang et al., 1997); however, we were unable to demonstrate
Ca2+-pumping activity because of high activity from the
Ca2+ antiporter (Vcx1).
Here we provide the first biochemical characterization, to our
knowledge, of a plant Ca+ pump functionally expressed in
yeast. We show that ECA1 encodes a high-affinity plant
Ca2+ pump that is blocked by cyclopiazonic acid. ECA1
shares many similarities with animal SER-type Ca2+-ATPases;
however, it is unique in its insensitivity to thapsigargin. We also
demonstrate that a yeast triple mutant provides a powerful expression
system with which to study individual Ca2+ pumps from
heterologous systems.
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MATERIALS AND METHODS |
Yeast Strain, Plasmid, and Growth Medium
Yeast (Saccharomyces cerevisiae) strain K616
(MATa pmr1::HIS3 pmc1::TRP1
cnb1::LEU2, ura3; Cunningham and Fink, 1994) is often referred to as the triple mutant. The full-length cDNA of the ECA1 gene (accession no. U96455) from Arabidopsis was
constructed into the yeast expression vector p426Gal1 under the control
of the Gal-inducible promoter (Liang et al., 1997). The mutant K616 was
transformed with this construct or the empty vector using the lithium
acetate method (Chen et al., 1992). Transformants were selected on
SC-URA. The growth medium consisted of 6.7 g/L yeast nitrogen base
without amino acids, 2 g/L drop-out mixture without uracil, and 2% Gal
(Rose et al., 1990).
Yeast Growth
To measure the growth of mutant K616 strains transformed with
either ECA1 or with vector alone, cells at the
late-log phase were harvested by centrifugation and suspended in 10 mL of SC-URA that contained 1 mM Ca2+.
The cell suspension was used to inoculate 20 mL of SC-URA (pH 6.2) to
an initial A600 of 0.01. Either 10 mM Ca2+ or varying concentrations of
EGTA was added to the medium to control free-Ca2+
levels. Growth at 30°C was monitored by the change in
A600 of 0.8-mL samples for 2 d.
Isolation of Membrane Vesicles
Vesicles were isolated after cell disruption using the glass-bead
method (Liang et al., 1997) with some modification. Transformants were
inoculated into 25 mL of SC-URA and incubated overnight at 30°C. The
seed culture was diluted into 250 to 300 mL of SC-URA/Gal and incubated
until the A600 reached 1.5 to 2.0. Cells
were pelleted at 4000g for 5 min, washed with 10 mL of
distilled water, and pelleted. To isolate vesicles for transport
studies, 2 mM MgCl2 was included in
all of the solutions to facilitate separation of the ER from the
vacuolar vesicles (see below). The cell pellet was suspended in 10 mL
of glass-bead buffer and pelleted. The glass-bead buffer consisted of
10% Suc, 25 mM Hepes-BTP, pH 7.5, 2 mM
MgCl2, 2 mM DTT, and 1 mM
EGTA.
Typically, 3 to 4 mL of cells was resuspended in 1 volume of glass-bead
buffer plus 1 mM PMSF, 10 mM benzamidine, 5 µg/mL pepstatin, 5 µg/mL leupeptin, and 0.5% BSA, and split into
two Corning tubes (50 mL). An equal volume of glass beads (Sigma) was
added and the mixture was vortex mixed four times for 30 s each.
The lysate was centrifuged at 5,000g for 5 min and the
supernatant was saved. The pellet was suspended in 1 volume of
glass-bead buffer plus protease inhibitors, vortex mixed, and
centrifuged as described above. Then, 2 to 3 mL of the pooled
supernatant was layered onto a step gradient containing 6 mL each of
25% and 45% Suc in 20 mM Hepes-BTP (pH 7.0), 1 mM DTT, 2 mM MgSO4, 0.2 mM PMSF, and 5 mM benzamidine, and centrifuged
(model SW 28 centrifuge, Beckman) at 108,000g for 2 h.
Membranes at the 26%/45% Suc interface were collected and diluted 6- to 8-fold in a suspension solution containing 25 mM
Hepes-BTP (pH 7.0), 1 mM DTT, 2 mM
MgSO4, and protease inhibitors. After the sample
was centrifuged at 108,000g for 50 min, the pellet was
suspended in the same solution and stored at 
80°C. The protein
concentration was determined with the Bio-Rad reagent.
To determine the distribution of ECA1 in yeast membranes, microsomes
were isolated in the presence or absence of Mg2+.
About 0.5 mL of cells from 50 mL of overnight culture was suspended in
1 volume of glass-bead buffer with either 2 mM
MgSO4 or 2 mM EDTA. The glass-bead
buffer included 0.5 mM PMSF, 2 mM benzamidine, 5 µg/mL pepstatin, 5 µg/mL leupeptin, and 0.5% BSA. Cells were disrupted with the buffer as described above. The lysate was
centrifuged at 5,000g for 5 min and the supernatant was
saved. The pellet was suspended in glass-bead buffer, vortexed, and
centrifuged as described above. The supernatants were pooled and
pelleted at 108,000g for 50 min. The microsomal pellet was
resuspended in 0.8 mL of the above solution without BSA and layered
onto a step gradient with 1.2 mL each of 12%, 15%, 18%, 21%, 24%,
27%, 30%, 33%, 36%, 39%, 42%, and 45% Suc. The Suc solutions
contained 25 mM Hepes-BTP, pH 7.0, 1 mM DTT,
0.1 mM PMSF, and 2 mM benzamidine with either 2 mM MgSO4 or 2 mM EDTA.
After the sample was centrifuged at 110,000g for 16 h,
0.75-mL fractions were collected and stored at 80°C.
45Ca2+ Uptake
Ca2+ uptake into membrane vesicles was
measured by the filtration method. Typically, transport was initiated
with 3 mM ATP in a reaction mixture (250 µL) containing
250 mM Suc, 25 mM Hepes/BTP (pH 7.0), 10 mM KCl, 0.4 mM NaN3, 3 mM MgSO4, 100 µM EGTA,
and 10 µM 45CaCl (3000 Ci/mmol,
NEN-Dupont) so the final specific activity was 1 to 2 µCi/2.5 nmol
Ca2+ per reaction. Under these conditions, the
calculated free-Ca2+ concentration is about 0.1 µM (Bers et al., 1994). For measuring 
pH-independent
Ca2+-pumping activity, 0.5 µM
bafilomycin A1 and 5 µM gramicidin D were routinely
included. After incubation at 22°C, 220 µL was run through a filter
(0.45-µm pore size, Millipore) moistened with a rinse solution
containing 250 mM Suc, 2.5 mM Hepes-BTP (pH
7.0), and 0.2 mM CaCl2. The filter
was washed with 5 mL of a cold rinse solution. The
45Ca2+ radioactivity
associated with the filter was determined by liquid-scintillation counting. Active transport was determined as the difference between activity in the presence and absence of ATP, and is expressed as
nanomoles of Ca2+ per milligram of protein. All
inhibitors were preincubated with membranes at 22°C for 15 min before
the reaction was started. To determine the
Km for Ca2+, the
reaction mixture contained 500 µM EGTA and various
amounts of Ca2+ to give the desired range of the
free-Ca2+ concentration (10 nM to 2 or 3 µM).
Electrophoresis, Immunostaining, and Calmodulin Overlay
These procedures were described previously (Hwang et al., 1997;
Liang et al., 1997) and are briefly described in the figure legends.
After SDS-PAGE, proteins were transferred to Immobilon-P membranes
(Millipore) in 25 mM Tris, pH 8.3, 192 mM Gly,
and 15% methanol. To determine the calmodulin binding, the blot was
blocked for 1 h with 1% BSA in TBS/CaMg (50 mM Tris,
pH 7.5, 0.2 M NaCl, and 50 mM
MgCl2, with or without 0.5 mM
CaCl2). Then the blot was incubated with 100 ng/mL biotinylated calmodulin in TBS/CaMg for 2 h at 22°C
and washed twice in TBS/CaMg plus 0.05% Tween 20. After the sample was
incubated with streptavidin conjugated to alkaline phosphatase, color
was developed with 5-bromo-4-chloro-3-indoyl phosphate and nitroblue
tetrazolium (Sigma). To detect nonspecific binding, electroblotted
proteins were incubated with biotinylated calmodulin in the
presence of 1 mM EGTA. Bovine calcineurin (Sigma) was used
as a positive control.
Chemicals
Erythrosin B, cyclopiazonic acid, and gramicidin D were obtained
from Sigma, and thapsigargin was purchased from LC Service Co. (Woburn,
MA). Bafilomycin A1 was a gift from Dr. Karlheinz Altendorf (University
of Osnabrueck, Germany). All other chemicals were of a reagent
grade.
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RESULTS |
An Arabidopsis Ca2+-ATPase ECA1 Increased Yeast Mutant
K616 Growth on Ca2+-Depleted Medium
The yeast mutant K616 defective in both the Golgi (Pmr1) and the
vacuolar Ca2+ (Pmc1) pumps was unable to grow on
SC-URA plates containing 10 mM EGTA; therefore, we tested
the growth rates of mutants expressing an Arabidopsis
Ca2+-ATPase, ECA1, as a function of
Ca2+ concentration. Although the control K616
mutant transformed with vector alone had a doubling time of 5 h in
medium containing 1 to 10 mM Ca2+,
growth was severely retarded as the free-Ca2+
concentration was lowered to 0.3 µM by 20 mM
EGTA (Fig. 1A). In contrast, mutants
transformed with the ECA1 gene had a doubling time of about
5 h when external Ca2+ was <1
µM (Fig. 1B). The initial free-Ca2+
concentration was estimated with the Max-Chelator program (Bers et al.,
1994) based on the amount of EGTA added to the medium, which contained
1 mM Ca2+. At 31 h after
inoculation, the relative density of ECA1-transformed mutants was 10- to 20-fold higher than that of the control mutants grown in medium containing approximately 0.3 µM
Ca2+. The differential growth rate was
consistently observed even when the medium pH was buffered to 6.0 (not
shown). Therefore, expression of an Arabidopsis
Ca2+-ATPase enhanced yeast mutant growth in
submicromolar levels of Ca2+ to a rate nearly
comparable to that of wild-type strains (not shown).
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| Figure 1.
ECA1 enhanced growth of yeast
mutant K616 in Ca2+-depleted medium. A and
B, Time course of yeast growth. Mutants transformed with vector (A) or
ECA1 (B) were suspended in SC-URA (pH 6.2) to a final
A600 of 0.01 and incubated at 30°C. The
medium was either used directly ( ) or supplemented with 10 mM CaCl2 ( ) or 2 ( ), 5 ( ), 10 ( ),
or 20 ( ) mM EGTA. The turbidity of the culture was
measured at 600 nm over time. C, Effect of free-Ca2+
concentration on growth of mutant K616 harboring vector control ()
or ECA1 (). A600 was
determined after 31 h of growth.
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To determine the subcellular location of ECA1 in yeast, microsomes were
isolated from ECA1-transformed mutants and fractionated by a
Suc-density gradient. The fractions were analyzed by SDS-PAGE, blotted,
and immunostained with a polyclonal antibody against the carboxyl
terminus of ECA1. In the presence of EDTA, ECA1 was broadly distributed
with an apparent peak at approximately 32% Suc. When
Mg2+ was present during cell isolation and in
gradient fractionation, ECa1p was concentrated at 38% to 40% Suc
(Fig. 2). As EDTA chelates Mg2+ and dissociates ribosomes from the ER, the
shift in ECA1 suggested that this polypeptide was associated with the
ER. This interpretation agrees with other studies in which yeast ER was
found at 40% Suc (Antebi et al., 1992). However, overexpression of
this protein could result in localization to other endomembranes, such
as the Golgi. To investigate the transport properties of ECA1, vesicles were routinely isolated from the interface of a 26%/45% Suc step gradient containing Mg2+.
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| Figure 2.
ECA1 was associated with ER vesicles from yeast.
Microsomes were isolated from the ECA1-transformed
mutant and fractionated with a Suc gradient containing 2 mM
EDTA (top) or 2 mM MgCl2 (bottom). Proteins
(1-5 µg/lane) were separated by 7% SDS-PAGE, blotted, and
immunostained with polyclonal antibodies against ECA1 (1:10,000).
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The Control K616 Mutant Has High
H+/Ca2+Antiport Activity
We first tested for background Ca2+
transport in vesicles isolated from the control mutant K616 transformed
with vector alone. At a free-Ca2+ concentration
of approximately 0.1 µM, ATP increased
45Ca2+ associated with
vesicles. The Ca2+ was released by the ionophore
A23187, indicating that it had accumulated against a concentration
gradient. Transport was resistant to sodium vanadate, a P-type
cation-pumping ATPase inhibitor (Nechay, 1984), but was decreased by
gramicidin D, bafilomycin A1, or both (Fig.
3). Since bafilomycin specifically
inhibits proton pumping by the vacuolar H+-ATPase
and gramicidin dissipates proton gradients, the results demonstrated
that more than 95% of Ca2+ transport was pH
dependent (Fig. 3). Thus, nearly all of the Ca2+
uptake was driven by the
H+/Ca2+-antiport activity
of the Vcx1 (Cunningham and Fink, 1996). Either gramicidin or
bafilomycin alone inhibited 80% to 90% of the pH-dependent Ca2+ uptake (data not shown); however, both
compounds were needed to achieve maximum inhibition of antiport
activity.
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| Figure 3.
Active 45Ca2+ uptake into
vesicles of control mutant is pH dependent. Microsomal vesicles were
isolated from the vector-transformed K616 strain. Uptake was assayed in
a mixture containing 0.1 µM free Ca2+. A,
Total uptake. Activity was assayed in the absence (ATP, ) or
presence (+ATP) of 3 mM ATP with or without inhibitors
(). When added, the concentrations of sodium vanadate (Van, ) or
gramicidin D and bafilomycin A1 (G + Baf  ) were 200 or 5 and 0.5 µM, respectively. B, pH-dependent (pH, )
and vanadate-sensitive (Van, ) Ca2+ transport. pH
or (G + Baf) refers to the difference between total activity
(+ATP) and that measured with bafilomycin and gramicidin (+ATP + G + Baf). Van refers to the difference between total activity (+ATP) and
that measured in the presence of vanadate (+ATP + Van). The arrows
indicate addition of 1 µM ionophore A23187.
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The pH-dependent Ca2+ transport had an
apparent affinity for Ca2+ of 350 ± 50 nM (n = 5; Fig.
4), which is lower than the published Km for Ca2+ of 10 to
100 µM reported for wild-type yeast (Ohsumi and Anraku, 1983; Dunn et al., 1994). A small component (<10%) of the
vesicle-associated Ca2+ appeared to be vanadate
sensitive when free [Ca2+] was >0.1
µM; however, this component was mainly due to
ATP-dependent binding because the Ca2+ was not
released by the ionophore A23187 (not shown).
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| Figure 4.
Ca2+ affinity of pH-dependent
Ca2+ transport. Vesicles were isolated from mutant K616
transformed with vector. Left, Uptake was determined at 40 s after 3 mM ATP was added to a reaction medium containing
10 nM to 3 µM free Ca2+.
pH-dependent Ca2+ transport (pH, ) was determined
as activity that was inhibited by 5 µM gramicidin and 0.5 µM bafilomycin. Vanadate-sensitive Ca2+
transport refers to activity inhibited by 200 µM sodium
vanadate (Van, ). Right, Lineweaver-Burk plot. pH-dependent
uptake has a Km for Ca2+ of 350 ± 50 nM (n = 5).
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ECA1-Catalyzed Ca2+-Pump Activity Is pH Independent
To determine whether ECA1 encoded a functional
Ca2+ pump, membrane vesicles were isolated from
ECA1-transformed mutants and assayed for
45Ca uptake. In contrast to the findings shown in
Figure 3, sodium vanadate consistently inhibited ATP-driven
Ca2+ uptake partially (Fig.
5A). Furthermore, a component (50%) of the Ca2+ accumulated was resistant consistently
to a combination of 5 µM gramicidin and 0.5 µM bafilomycin A1 (Fig. 5). Thus,
the transport component that was pH independent and vanadate
sensitive resembled activity from a Ca2+ pump.
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| Figure 5.
Vanadate-sensitive and pH-independent
Ca2+-pumping activity. Vesicles were isolated from the
ECA1-transformed mutant K616. 45Ca uptake
was assayed as described in Figure 3. A, Total uptake. Uptake was
determined without (ATP, ) or with ATP in the absence (+ATP, )
or presence of 200 µM sodium vanadate (+Van, ) or 5 µM gramicidin and 0.5 µM bafilomycin (G + Baf, ). B, pH-independent () and vanadate-sensitive (Van,
) uptake were similar initially. The initial rate was approximately
4 nmol mg 1 protein min1. The arrows
indicate the addition of 1 µM ionophore A23187.
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Ca2+ uptake driven by the antiport, but not by
the pump, was enhanced 4-fold and 3-fold by 10 mM potassium
oxalate and 10 mM potassium phosphate, respectively (Table
I). Oxalate stimulation of
Ca2+ uptake is thought to be caused by formation
of Ca2+ oxalate precipitate inside the vesicles,
thus decreasing the magnitude of the Ca2+
chemical gradient. Formation of Ca2+ phosphate
precipitate in vesicles will result in a similar stimulatory effect of
Ca2+ uptake. The differential stimulation of
anions suggested that an oxalate carrier colocalized to the same
vesicle membrane as the antiporter and that the pump, in spite of
overexpression, resided on another membrane that was not permeable to
oxalate or phosphate.
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Table I.
Potassium oxalate and potassium phosphate stimulated
Ca2+/H+-antiport but not Ca2+-pump
activity
Net ATP-dependent 45Ca2+ uptake at 6 min was
determined with 0.1 µM Ca in the reaction mixture as
described in Figure 5. Uptake was measured in the absence (Total) or
presence (Pump) of 5 µM gramicidin and 0.5 µM bafilomycin. Antiport activity was determined as the
difference between "total" and "pump" activity. Potassium
oxalate or potassium phosphate was added to a final concentration of 10 mM. Data represent the averages of two experiments.
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Although the initial rate of Ca2+ uptake by the
pump (4 nmol mg1 min1)
was 3- to 4-fold higher than that of the antiporter (1 nmol mg1 min1), net uptake
by antiport activity was 3- to 4-fold higher than that by the pump.
This difference could be due to one or more of the following: (a)
differential driving force, (b) differential transport rate, or (c)
differential Ca2+ leakage from the compartments
via channels. These results further support the idea that the pump and
antiport reside in separate compartments.
Although pump activity could be determined as either pH-independent
or vanadate-inhibitable Ca2+ activity, net uptake
of the vanadate-sensitive component declined over time (Fig. 5B). It is
possible that the high-capacity Vcx1 competed with the pump for free
Ca2+ in the absence of bafilomycin and thus
depleted the free-Ca2+ concentration in the
medium. Therefore, subsequent ECA1-pump activity was
measured as pH-independent transport in the presence of gramicidin
and bafilomycin A1.
High Affinity for Ca2+ and ATP
The initial rate of pumping was measured as bafilomycin- and
gramicidin-resistant Ca2+ uptake at 40 s
when uptake was nearly linear with incubation time (Fig. 5A).
Free-Ca2+ concentration was controlled by varying
the total Ca2+ added in the presence of 0.5 mM EGTA to give a final free-ion concentration ranging from
10 to 2000 nM (not shown). Surprisingly, the average
Km for Ca2+
determined from five independent experiments was 30 ± 10 nM (Fig. 6). Thus, this
ER-type Ca2+-ATPase has a high affinity for
Ca2+ and reached a maximum velocity when
Ca2+ was approximately 0.1 µM.
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| Figure 6.
ECA1 catalyzed high-affinity
Ca2+-pumping activity. Ca2+ transport into
vesicles isolated from the ECA1-transformed mutant K616
was performed as described in Figure 4, except that the
free-Ca2+ concentration was buffered from 10 to 70 nM. Lineweaver-Burk plot (inset) showed an apparent
Km for Ca2+ of 30 ± 10 nM (n = 5).
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The initial rate of Ca2+ transport showed a
biphasic dependence on ATP (Fig. 7),
which is analogous to animal Ca2+ pumps. All
assays were conducted at 40 s, when the ATP concentration had not
changed significantly. These data are consistent with a model of two
ATP-binding sites with Km values of 20 and
235 µM, respectively. GTP also energized
Ca2+ pumping; however, we did not detect evidence
for biphasic kinetics. The affinity of the pump for GTP was low, and
the estimated Km for GTP was greater than
1.5 mM.
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| Figure 7.
High affinity for ATP. Vesicles were isolated from
ECA1-transformed mutants for ATP- or GTP-dependent
45Ca transport. Reaction mixtures contained 0.1 µM free Ca2+, 5 µM gramicidin,
0.5 µM bafilomycin, and 25 µM to 3 mM ATP () or GTP (). Uptake was measured at 40 s. Apparent ATP Km values were 20 and 235 µM. The results are the averages of three experiments.
conc., Concentration.
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Inhibitors of the ECA1 Pump
The initial rate of ECA1-catalyzed Ca2+
uptake was inhibited 50% by 1.5 µM sodium orthovanadate
(Fig. 8A), a diagnostic inhibitor of
P-type ATPases. Erythrosin B at about 0.5 µM inhibited
Ca2+-pump activity by 50% (Fig. 8B). Erythrosin
B is a halogenated derivative of fluorescein that could modify a Lys
residue close to the ATP-binding site, causing inhibition of ATP
binding (Mignaco et al., 1996).
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| Figure 8.
Inhibition of the ECA1 pump by
vanadate and cyclopiazonic acid but not by thapsigargin. Vesicles were
preincubated with each inhibitor or DMSO for 15 min at room temperature
in the absence of ATP. To start the reaction, ATP was added to a
mixture containing 0.1 µM free Ca2+ and
various concentrations of sodium vanadate (A), erythrosin B (B),
cyclopiazonic acid (C), or thapsigargin (D). The transport was stopped
at 40 s. Control activity (100%) was 1.8 nmol Ca2+
mg1 protein min1. Data are the averages of
three experiments. I50, Inhibitor concentration for 50%
displacement.
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Cyclopiazonic acid, an indole tetramic acid, has been reported to be a
specific inhibitor of SER-type Ca2+-ATPase
(Seidler et al., 1989). Pump activity from ECA1 was blocked 50% by 3 nmol cyclopiazonic acid mg1 protein (Fig. 8C).
This value is comparable to the concentration required to block SERCA.
However, thapsigargin, a potent and specific inhibitor of SER-type
Ca2+ pumps, had no effect on Ca2+
transport at concentrations up to 3 µM (or 66 nmol
mg1 protein; Fig. 8D).
No Effect by Calmodulin
Because calmodulin-stimulated Ca2+ transport
has been detected in the ER fraction from carrot cells (Hsieh et al.,
1991; Hwang et al., 1997), we tested the effect of calmodulin on pump
activity. Net Ca2+ uptake at 5 min was 0.65 nmol
mg1 protein with or without 1 µM
calmodulin, suggesting that calmodulin did not stimulate this pump. To
test whether ECA1 bound calmodulin, membranes were isolated and the
proteins were separated by SDS-PAGE, blotted, and probed with
biotinylated calmodulin (Fig. 9). As a
positive control, we showed that calmodulin bound the 60-kD calcineurin
A subunit in a Ca2+-dependent manner (Fig. 9,
lanes 6 and 9). ECA1 was detected by immunostaining only in
ECA1-transformed, but not in control, mutants (Fig. 9, lanes
1 and 2). However, calmodulin failed to bind any protein at
approximately 116 kD from either membrane preparation. Thus, the
ER-type Ca2+ pump encoded by ECA1 was
not directly regulated by calmodulin.
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| Figure 9.
ECA1 did not bind calmodulin. Membranes were
isolated from cells transformed with either vector alone (Vec.) or
ECA1. Vesicle protein (5 µg) from the 25%/45% Suc
interface was analyzed by SDS-PAGE, blotted onto an Immobilon-P
membrane, and probed with an antibody against ECA1 (lanes 1-3).
Another blot was probed with biotinylated calmodulin in the absence (+1
mM EGTA, lanes 4-6) or presence of (+Ca2+,
lanes 7-9) 0.5 mM Ca2+. The bovine calcineurin
A subunit (CNA; 0.2 µg protein/lane) is a calmodulin-binding
protein.
|
|
| |
DISCUSSION |
A Functional Plant Ca2+ Pump Expressed in
Yeast
Here we provide the first biochemical characterization, to our
knowledge, of a functional Ca2+ pump encoded by a
plant gene, ECA1. The pump properties were determined after
the gene was expressed in a yeast mutant strain, K616, that lacked both
the Golgi (Pmr1) and the vacuolar (Pmc1) Ca2+
pumps (Cunningham and Fink, 1994; Catty et al., 1997). The advantages of the triple mutant are: (a) the growth rate of the mutant on Ca2+-depleted medium provides an in vivo assay
for functional identification of active Ca2+
pumps, (b) the control mutant is devoid of any background
Ca2+-pump activity (Fig. 3) or
Ca2+-dependent phosphoproteins (Liang et al.,
1997), and (c) any heterologous gene encoding a
Ca2+ pump could be overexpressed with a strong
inducible promoter. Thus, the triple mutant provides an extremely
valuable expression system for determining the nature and
structure/function relationships of individual
Ca2+ pumps from other eukaryotes.
Despite these advantages, Ca2+-pump activity is
frequently masked by high
H+/Ca2+-antiport activity
in the triple mutant. Cunningham and Fink (1994, 1996) found that the
double mutant pmr1pmc1 was not viable unless calcineurin
function was disrupted. Evidence suggested that calcineurin acted as a
negative regulator of VCX1 in wild-type strains. Loss of
calcineurin function resulted in a triple mutant K616
(pmr1pmc1cnb1) that could tolerate high levels of
Ca2+, apparently by activating the
H+/Ca2+ antiport. Three
strategies were used to separate pump activity from the vacuolar
H+/Ca2+-antiport activity.
First, vesicles enriched in ECA1 were collected from a 26%/45% Suc
interface to minimize vacuoles at 22% Suc (Antebi and Fink,
1992; Sorin et al., 1997). Second, pump activity was assayed
after inhibition of
H+/Ca2+-antiport activity
with proton ionophore, gramicidin, and a specific vacuolar
H+-ATPase inhibitor, bafilomycin. Third, the
free-Ca2+ concentration was reduced to 0.1 µM to reduce antiport activity and to minimize
Ca2+ binding to membranes.
A High-Affinity Ca2+ Pump
The most striking feature of the ECA1 pump is its high affinity
for Ca2+. The pump showed a
Km for Ca2+ of about
30 nM (Fig. 6) relative to the
Km of 350 nM of the
H+/Ca2+ antiport. Transport
was dependent on both Mg2+ (data not shown) and
ATP (Fig. 5), and 1 to 3 mM was required for maximum pump
activity (Fig. 7). It is interesting that the ECA1 pump displayed two
affinities for ATP, 20 and 235 µM. In animal
Ca2+ pumps the high-affinity site of about 2 µM is the hydrolytic site, whereas the low-affinity site
ranging from 100 to 300 µM accelerates the reaction
(Schatzmann, 1989). It is not clear whether the two sites are separate
or whether one site changes in affinity in the reaction cycle.
Inhibition by Cyclopiazonic Acid but Not by Thapsigargin
Although sodium vanadate inhibited Ca2+
transport with a 50% inhibition of approximately 1.5 µM
(Fig. 8A), it had little or no effect on the initial rate of
phosphorylation (F. Liang, unpublished data). These results indicate
that vanadate does not block the early steps in the reaction cycle. The
reaction cycle of animal SERCA can move in the direction of ATP
synthesis. In this mode, vanadate inhibits the Pi-dependent formation
of E2P by competing for the
phosphate-binding site (Schatzmann, 1989). Thus, vanadate inhibits
Ca2+ transport, which depends on completion of
many reaction cycles.
However, the ECA1 pump is insensitive to thapsigargin (Fig. 8D), a
specific inhibitor of SERCA (Sagara and Inesi, 1991). It binds to SERCA
with a one-to-one stoichiometry and a subnanomolar affinity on the
third transmembrane segment (Norregaard et al., 1994). During turnover
thapsigargin slowly inhibits activity by binding to the
Ca2+-deprived intermediate at each cycle, leading
to total inactivation. Thus, in a 1-mL reaction mixture containing 1 µg of sarcoplasmic reticulum protein, 4 pmol of thapsigargin
(4 nM) will completely inhibit 4 pmol of the SERCA pump
(Sagara and Inesi, 1991). We have estimated the relative amount of the
Arabidopsis ER Ca2+-ATPase expressed in yeast
membranes. The steady-state level of phosphoenzyme formed at 300 nM ATP (when rate was near maximum) was 120 to 150 pmol
mg1 membrane protein (F. Liang, unpublished
data). With a molecular mass of 116 kD, the plant
Ca2+ pump represents at least 1.4% to 1.7% of
the membrane protein. Thus, the plant Ca2+ pump
is 15- to 20-fold overexpressed in yeast membranes relative to native
Ca2+-ATPases, which make up approximately 0.01%
of plant membranes (Chen et al., 1993). However, 3 µM
thapsigargin, which corresponds to 66 nmol mg1
membrane protein, did not inhibit ECA1 pumping. Thus, this
isoform of plant ER-type Ca2+ pump does not bind
to thapsigargin, although the third transmembrane segment of ECA1
shared 67% identity with the thapsigargin-interaction site of SERCA.
The difference between plant and animal ER-type Ca2+ pumps might be because thapsigargin is a
plant-derived sesquiterpene lactone.
It is interesting that the plant ECA1 pump is inhibited by another
specific SERCA inhibitor, cyclopiazonic acid, although the mode of
action is still unclear. Cyclopiazonic acid inhibited sarcoplasmic
reticulum Ca2+-ATPase stoichiometrically,
but the indole tetramic acid at 1000 nmol mg1
had no effect on the PM Ca2+-ATPase or other
ion-pumping ATPases (Seidler et al., 1989). Binding of
cyclopiazonic acid decreases the enzyme affinity for ATP 10-fold, and
recent studies suggested that cyclopiazonic acid and ATP do not compete
for the same binding site (Plenge-Tellechea et al., 1997). Both
phosphoenzyme formation and Ca2+ transport of the
plant ECA1 pump are inhibited with a 50% inhibition of approximately 3 nmol mg1 (Fig. 8C). Thus, binding of
cyclopiazonic acid could reduce the affinity of ECA1 for ATP and
consequently decrease the initial rate of phosphoenzyme formation
(Liang et al., 1997) and Ca2+ transport (Fig.
8C).
Function of ECA1 in Arabidopsis
It is interesting that the localization of ECA1 on the ER and
perhaps the Golgi of Arabidopsis plants (Liang et al., 1997) is similar
to its expression on the ER and on endomembrane compartments of yeast.
Furthermore, ECA1 is localized on membranes distinct from the vacuolar
H+/Ca2+ antiport in yeast
(Table I), suggesting that a potential ER retrieval signal, KXKXX, at
the carboxyl terminus of the plant pump is recognized in yeast. If so,
the function of the plant Ca2+ pump in yeast may
reflect in part its native role in plants. ECA1 could restore
pmr1 mutant growth on Ca2+-depleted
medium (Liang et al., 1997), indicating that it resembled the function
of the native yeast Golgi Ca2+ pump (Sorin et
al., 1997). However, ECA1 could pump other divalent cations, including
Mn2+, because expression of ECA1
restored the growth of a yeast pmr1 mutant on
Mn2+-containing medium (Liang et al., 1997). It
is interesting that plants treated with cyclopiazonic acid showed
aggregation of ER membranes (Busch and Sievers, 1993). Furthermore,
gravitropic responses are inhibited in cress roots treated with
cyclopiazonic acid (Sievers and Busch, 1992). After overexpression of a
rice ER-type Ca2+-ATPase, the GA3
requirement for 
-amylase induction and secretion in aleurone
protoplasts was bypassed (Chen et al., 1997). These results support the
idea that: (a) luminal Ca2+ supplied by ER
Ca2+-pumping ATPase could play a role in protein
processing and vesicle trafficking of the ER and Golgi (Beckers and
Balch, 1989), and (b) ER Ca2+ pumps modulate
cytoplasmic Ca2+ waves induced by various
stimuli. Thus, like yeast cells, the normal growth and development of
plant cells are dependent on active high-affinity
Ca2+ pumps on the ER and Golgi.
| |
FOOTNOTES |
1
This work was supported in part by Department of
Energy grant no. DE-95ER20200 and by Maryland Agricultural Experiment
Station grant no. MD-J-151 (to H.S.).
2
Present address: The Institute for Genomic
Research, 9712 Medical Center Drive, Rockville, MD 20850.
*
Corresponding author; e-mail hs29{at}umail.umd.edu; fax
1-301-314-9082.
Received April 17, 1998;
accepted July 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane.
PM, plasma
membrane.
SC-URA, synthetic complete medium minus uracil.
SER, sarcoplasmic/ER.
SERCA, SER Ca2+ ATPase.
| |
ACKNOWLEDGMENTS |
We would like to thank K.W. Cunningham (Johns Hopkins
University) and J.F. Harper (The Scripps Research Institute) for
providing yeast strains and an antibody against Arabidopsis ECA1/ACA3
protein, respectively.
| |
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Abstract
Introduction