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Maize cap1 Encodes a Novel SERCA-type Calcium-ATPase with a
Calmodulin-binding Domain*
Received for publication, February 22, 2000, and in revised form, April 17, 2000
Published, JBC Papers in Press, April 17, 2000, DOI 10.1074/jbc.M001484200
Chalivendra C. Subbaiah‡§ and Martin M. Sachs‡¶
From the ‡Department of Crop Sciences, University of Illinois, Urbana and the ¶United States Department of
Agriculture/Agricultural Research Service, Soybean/Maize Germplasm, Pathology and Genetics Unit,
Urbana, Illinois 61801
A cDNA (CAP1) isolated from maize roots shares se-
quence identity with genes encoding P-type Ca
2ⴙ
-AT-
Pases and restores the growth phenotype of yeast mu-
tants defective in Ca
2ⴙ
-pumps. CAP1 was transcribed
and translated in the yeast mutant. Furthermore, the
membrane-integrated product formed a Ca
2ⴙ
-depend-
ent phosphorylated intermediate and supported Ca
2ⴙ
transport. Although CAP1 shares greater sequence iden-
tity with mammalian “endoplasmic reticulum-type”
Ca
2ⴙ
-pumps, it differs from these genes by having fea-
tures of calmodulin (CaM)-regulated Ca
2ⴙ
-pumps. CAP1
from yeast microsomes bound CaM, and the CAP1-de-
pendent Ca
2ⴙ
transport in yeast was stimulated by CaM.
Peptides from the C terminus of CAP1 bound CaM. Anti-
CAP1 antibodies specifically recognized a maize micro-
somal polypeptide that also bound CaM. A similar
polypeptide also formed a Ca
2ⴙ
-dependent phosphoen-
zyme. Our results suggest that cap1 encodes a novel
form of CaM-regulated Ca
2ⴙ
-ATPase in maize. CAP1 ap-
pears to be encoded by one or two genes in maize. CAP1
RNA is induced only during early anoxia, indicating
that the Ca
2ⴙ
-pump may play an important role in O
2
-
deprived maize cells.
Previous studies have shown that an elevation of cytosolic
calcium ([Ca
2⫹
]
i
) precedes molecular and whole plant re-
sponses to oxygen deprivation in maize (1, 2). Furthermore,
anoxia-induced [Ca
2⫹
]
i
elevation in maize cells falls into two
distinct patterns, differing in their magnitude and kinetics (1).
Tight regulation of [Ca
2⫹
]
i
is essential to the proposed role for
Ca
2⫹
as a cellular messenger (3). The maintenance of [Ca
2⫹
]
i
at
submicromolar levels and regulation of spatio-temporal pat-
terns of Ca
2⫹
signals are mediated by various Ca
2⫹
transport-
ers, such as Ca
2⫹
-pumps and proton-coupled antiporters (see
e.g. Ref. 4). Hence, there has been a great interest in molecular
cloning of Ca
2⫹
transporters and characterizing their role in
Ca
2⫹
-mediated signaling pathways (5–9). As part of our anal-
ysis of the pathway and components of Ca
2⫹
-mediated anoxia-
signaling in maize, we initiated studies to identify Ca
2⫹
stores
(10) and transporters that may regulate the cytoplasmic Ca
2⫹
signal. Here, we report the isolation and characterization of a
cDNA clone (CAP1) that encodes a calmodulin-binding Ca
2⫹
-
ATPase in maize roots.
Ca
2⫹
-ATPases belong to two distinct classes, differing in
their size, sequence, cellular location, and regulation by cal-
modulin (CaM).
1
The “ER-type” pump (located on the ER mem-
branes) is a ⬃90-kDa polypeptide, lacks a CaM-binding do-
main, and is not dependent on CaM for its regulation. The
“PM-type” pump (located on the plasma membrane) is a 138-
kDa molecule, possesses a C-terminal CaM-binding region, and
is regulated by CaM. Homologs of ER-type as well as PM-type
Ca
2⫹
-pumps (but with the CaM-binding domain at the N ter-
minus) have been cloned from plants, recently (5–9). In maize,
a calmodulin-regulated Ca
2⫹
-pump, related in size and antige-
nicity to the animal PM-type Ca
2⫹
-ATPase, was purified from
endomembranes of young etiolated shoots (11–13). On the
other hand, biochemical evidence was presented for the exist-
ence of an intriguing ER-type pump with CaM-binding proper-
ties, in the enriched ER membranes of young etiolated maize
shoots (14). Our results indicate that cap1, by virtue of its
sequence identity to ER-type Ca
2⫹
-pumps and at the same time
possessing a C-terminal CaM-binding domain characteristic to
PM-type pumps, may encode a novel chimeric Ca
2⫹
-ATPase
such as the one implied by previous studies (14). In addition,
we have functionally characterized the CAP1 protein using a
yeast expression system and identified a putative cognate pro-
tein in maize microsomes. Furthermore, our expression analy-
sis suggests that the abundance of CAP1 mRNA is low in maize
roots and is mildly increased under anoxia.
EXPERIMENTAL PROCEDURES
Three-day-old dark-grown maize (Zea mays L., inbred B73Ht) seed-
lings were raised and anoxically treated, as described previously (2).
Yeast (Saccharomyces cerevisiae) strains W303-1A (MATa, leu2,
his3, ade2, trp1, ura3), pmr1 AA542 (MATa, pmr1:: HIS3, ade2, trp1,
ura3), pmc1 K605 (MATa, pmc1::TRP1, ade2, ura3), pmr2 K633
(MATa, pmr2:: HIS3, ade2, trp1, ura3), and triple mutant K616 (MATa,
pmr1:: HIS3, pmc1::TRP1 cnb1::LEU2, ura3) were generously provided
by Dr. Kyle Cunningham, The Johns Hopkins University. Wild type
and mutant strains were grown in standard YPD (15) except that
AA542 and K616 were supplemented with 10 mMCaCl
2
. Transforma-
tion was carried out according to Ref. 16, and the transformants were
selected on synthetic complete medium lacking uracil (SC-URA, Ref.
15).
Isolation and Analysis of cDNA Clones—A cDNA library constructed
in
ZAP vector (Stratagene) from the root tissue of 6-h anoxically
* This work was supported by National Research Initiative Compet-
itive Grants Program (NRICGP) Grant 96-35100-3143 from the United
States Department of Agriculture (to M. M. S. and C. C. S.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank
TM
/EBI Data Bank with accession number(s) AF096871.
§ To whom correspondence should be addressed: Dept. of Crop Sci-
ences, University of Illinois, Urbana, IL 61801. Tel.: 217-333-9743; Fax:
217-333-6064; E-mail: subbaiah@uiuc.edu.
1
The abbreviations used are: CaM, calmodulin; ER, endoplasmic
reticulum; PM, plasma membrane; PCR, polymerase chain reaction;
SC-URA, synthetic complete medium lacking uracil; SERCA, sarco/
endoplasmic reticulum calcium-ATPase; RACE, rapid amplification of
cDNA ends; HRP, horseradish peroxidase; MOPS, 4-morpholinepro-
panesulfonic acid; DTT, dithiothreitol; TM, transmembrane; BTP, 1,3-
bis[tris(hydroxymethyl)methylamino]propane; Os, Oryza sativa L.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 28, Issue of July 14, pp. 21678–21687, 2000
Printed in U.S.A.
This paper is available on line at http://www.jbc.org21678
by guest on October 31, 2015http://www.jbc.org/Downloaded from
treated 3-day-old maize cv. B73 seedlings (17) was screened using a
partial cDNA clone LCA1 that encodes a putative Ca
2⫹
-ATPase in
tomato (9). Fifteen positive clones were obtained by screening 2 ⫻10
5
plaques, and the one containing the longest insert (3.3 kilobase pairs,
CAP1) was sequenced using dideoxy chain termination method (United
States Biochemical Corp.). Sequence comparison and analyses were
carried out using BLAST search program available in the public domain
and MacDNASIS Pro program from Hitachi Software Engineering
America.
Cloning and Sequencing of 5⬘-RACE-PCR Products—A 22-base
primer derived from the 5⬘-end of CAP1 cDNA (20 bases inside) was
32
P-end-labeled and used to reverse transcribe the missing 5⬘-end of
CAP1 mRNA from maize root total RNA preparations. Precise products
that could later be PCR-amplified were obtained only at priming tem-
peratures ⬎65 °C, using the thermostable reverse transcriptases (Ther-
moScript RT, Life Technologies, Inc.; or Carboxydothermus hydrogeno-
formans polymerase, Roche Molecular Biochemicals). The primer
extension products were separated on 6% acrylamide-urea sequencing
gels.
32
P-Labeled 10 and 100 base ladders were co-run to size the
products. The 140-base-long product was eluted, PCR-amplified using a
hot-start method (PLATINUM Taq polymerase, Life Technologies,
Inc.), and cloned into Topo-TA vector (Invitrogen).
Expression of CAP1 Product in Yeast and Complementation of Yeast
Mutants—The entire open reading frame of CAP1 and a part of the
3⬘-untranslated region were PCR-amplified from the original cDNA
clone. An initiation codon at the 5⬘-prime and EcoRI sites at both
termini were introduced during amplification. The product was ligated
into the EcoRI site of a yeast expression vector p426Gal1 between the
galactokinase (Gal1) promoter and CYC1 termination sequence. The
junctions of the recombinant plasmid was sequenced to confirm the
orientation. The plasmid with correct orientation of the insert was used
to transform wild type and mutant yeast strains, and the transformants
were selected for uracil prototrophy on SC-URA plates. The Ura
⫹
col-
onies were used for complementation studies.
Overexpression of CAP1 Fragments in Escherichia coli and Purifica-
tion of Recombinant Proteins—Two fragments of CAP1, one that en-
codes a major part of the central hydrophilic loop (i.e. residues 342–576,
with a calculated molecular mass of 25 kDa) and the other from the
C-terminal region comprising residues from 947 to 1038 (⬃14 kDa),
were PCR-amplified and cloned into an expression vector pQE-30 (Qi-
aExpress, Qiagen). The vector contains an N-terminal histidine tag
downstream to the T5 promoter and the Lac operator. The chimeric
plasmids were transformed into appropriate hosts, and expression was
induced by isopropyl-1-thio-

-D-galactopyranoside. The recombinant
proteins were purified using a nickel-affinity column according to man-
ufacturer’s protocols (Qiagen). The expression of the C-terminal region
was limited by its apparent toxicity to E. coli. The affinity purification
of this peptide on nickel columns was also not successful, since a large
number of host proteins co-eluted with the recombinant product due to
hydrophobic interactions. However, the peptide could be purified to
homogeneity using CaM-Sepharose chromatography (see “Results”). A
calmodulin cDNA isolated from roots of maize inbred B73Ht seedlings
(Ref. 18, gift from Dr. G. Feix, Germany) was PCR-amplified and
subcloned into pQE-30. The clone was overexpressed, and the recombi-
nant CaM was purified to homogeneity using the QiaExpress system.
The identity and orientation of all the cloned DNA fragments was
confirmed by sequencing.
CaM Mobility Shift Assays—Synthetic peptides identical to parts of
the CAP1 C terminus were made using an Applied Biosystems Peptide
Synthesizer (Perkin-Elmer, Applied Biosystems Division) at the Pep-
tide Synthesis Facility of the University of Illinois, Urbana. Purity and
sequence of the peptides were verified by mass spectrometry, high
pressure liquid chromatography, and microsequencing. The peptides
were chosen based on their secondary structure (mean hydrophobicity
and the hydrophobic moment of axial helical projections). The ability of
the peptides to interact with calmodulin was studied using gel mobility
shift assays (19). Maize recombinant calmodulin or calmodulin purified
from maize roots was used for the assay. CaM was purified to homoge-
neity from maize roots as described (20).
Circular Dichroism Spectroscopy of Calmodulin and CAP1 Pep-
tides—The binding of CAP1 synthetic peptides to calmodulin was also
measured using CD spectra. Spectra of maize recombinant calmodulin
(3
M) and different molar equivalents of peptides were obtained in a
buffer containing 5 mMTris-Cl, 1 mMCaCl
2
(or2mMEGTA), 1 mMDTT
at pH 7.2 using a Jasco J-720 model spectropolarimeter (Jasco Inc.).
The signal was recorded using a 1-cm path length, a sensitivity of 20
millidegrees, a resolution of 0.5 nm, and at a scan speed of 20 nm/min.
The results presented are averages of three separate scans.
Purification of CaM-binding Proteins from Yeast and Maize Root
Microsomes—Microsomal proteins were prepared from yeast cells or
maize roots of 3-day-old dark-grown seedlings, and CaM-binding pro-
teins were purified using affinity chromatography essentially as de-
scribed (13). Briefly, detergent (1% Triton X-100 in 25 mMMOPS-BTP,
pH 7.5, 0.3 Msucrose, 0.5 MNaCl, 2 mMATP, 5 mMCaCl
2
, 0.1%
phospholipid, 2 mMDTT, 2 mMMgCl
2
and protease inhibitors)-solubi-
lized microsomal proteins were loaded on a 1- or 2-ml column of CaM-
Sepharose. The column was washed in 50 volumes of column buffer (25
mMMOPS-BTP, pH 7.5, 0.3 Msucrose, 0.5 MNaCl, 0.4 mMATP, 5 mM
CaCl
2
, 0.05% phospholipid, 0.05% Triton X-100, 1 mMDTT, and prote-
ase inhibitors), and CaM-binding proteins were eluted in a buffer con-
taining 25 mMMOPS-BTP, pH 7.5, 0.3 Msucrose, 10 mMEGTA, 1 mM
DTT, and protease inhibitors. For use in phosphoenzyme essays, 0.05%
Triton X-100 and 0.1% phospholipid was included in the elution buffer
and immediately brought up to 20 mMCaCl
2
. Single large scale prep-
arations of CaM-binding proteins were used to analyze polypeptide
composition, immunoassays with CAP1 antisera, phosphoenzyme for-
mation, and gel blot overlay with HRP-labeled CaM (detailed below).
Analysis of Phosphorylated Intermediate—Yeast microsomes pre-
pared as described (7) or maize root microsomal proteins fractionated on
CaM-Sepharose columns were used for the analysis of phosphorylation
intermediate. The reaction mixture (0.2 or 0.6 ml) contained 5–15
gof
protein, 10 nM[
␥
-
32
P]ATP (10
Ci), 1 mMEDTA, 0.5 mMEGTA, 100 mM
KCl, 25 mMHEPES-BTP, pH 6.0. The effects of Ca
2⫹
and La
3⫹
were
tested by adding CaCl
2
and LaCl
3
to give a final free ion concentration
of 100 and 50
M, respectively, as estimated by the MAXCHELATOR
program (21). The reaction was terminated, and proteins were resolved
in acidic phosphate gels (22, 23).
Ca
2⫹
Transport Assay with Yeast Membrane Vesicles—Membrane
isolation from K616 cells transformed with vector alone or with pCAP1
and the measurement of
45
Ca uptake were carried out, essentially as
described by Liang and Sze (24). The transport buffer contained 250 mM
sucrose, 25 mMHEPES-BTP, pH 7.5, 10 mMKCl, 3 mMMgSO
4
, 0.4 mM
sodium azide, 100
MEGTA, 5
Mgramicidin, and 10
M
45
CaCl (ICN).
The final specific activity was 2
Ci/10 nmol Ca
2⫹
per ml. Under the
conditions of pH, temperature, ATP, EGTA, and total calcium of our
assay, the calculated free Ca
2⫹
concentration varied between 10 nMand
50
M(21). Membrane vesicles equivalent to 15
g of protein were used
in 0.25 ml of reaction mix. Uptake was initiated by the addition of 3 mM
ATP and incubated at 25 °C for up to 20 min. The reaction mixture was
spotted on pre-wet GS filters (Millipore) under vacuum, and the filters
were washed in ice-cold rinse buffer containing 250 mMsucrose, 25 mM
HEPES-BTP, pH 7.5, and 200
MCa
2⫹
. When used, the calmodulin
inhibitor W7 was added at a final concentration of 100
Mand the Ca
2⫹
ionophore A23187 at 1
M.
Antibody Generation and Protein Gel Blot Assays—Monoclonal anti-
sera were raised against the 24-kDa central hydrophilic loop expressed
in E. coli. Calmodulin-binding proteins purified from maize microsomes
were concentrated by trichloroacetic acid precipitation and resolved in
8 or 6–12% gradient SDS-acrylamide gels. Gels were stained in silver or
Coomassie Blue. Proteins resolved in adjacent lanes were used for
immunoblot analysis. Protein transfer onto polyvinylidene difluoride
(Bio-Rad) and antibody incubations were done as described previously
(25).
Preparation of Horseradish Peroxidase-coupled CaM (HRP-CaM)
and CaM Overlay Assays—Maize recombinant calmodulin was conju-
gated to activated horseradish peroxidase (Pierce) by primary amine
coupling and was used to probe CaM-target interactions. The eluant
from CaM-Sepharose column was concentrated using trichloroacetic
acid precipitation. Proteins were separated on SDS-acrylamide gels and
blotted onto a polyvinylidene difluoride membrane. Subsequent block-
ing, incubation with HRP-CaM, and washings of the blot were carried
out as described (26). The peroxidase signal was visualized by
chemiluminescence.
DNA and RNA Gel Blot Hybridization—Maize genomic DNA was
isolated, digested with appropriate enzymes, and subjected to DNA gel
blot analysis according to standard protocols (27). Fragments from
three different regions of CAP1 clone, namely the 5⬘-end, sequence
coding for the central hydrophilic loop, and the 3⬘-end, were used as
probes. The hybridization and washings were done at moderate or high
stringency. Yeast mutant K616 cells transformed with p426Gal1 or
pCAP1 in SC-Ura medium containing glucose or galactose as the carbon
source. CaCl
2
at5mMwas added to support growth of pGal1 trans-
formed cells. Total RNA was isolated from 250-ml cultures grown for
20 h. RNA gel blot analysis was carried out as described previously (2).
Maize cap1 Encodes a Novel SERCA-type Calcium-ATPase 21679
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RESULTS
cap1 Encodes a Putative P-type Ca
2⫹
-ATPase—The clone,
CAP1, was isolated by screening a cDNA library made from the
root tissue of anoxic maize seedlings, with a tomato cDNA
encoding an ER-type Ca
2⫹
-ATPase (LCA1, 9). As shown in Fig.
1, the full-length CAP1 sequence can be translated into a
polypeptide of 1,049 amino acids with a calculated molecular
mass of 113,099 Da (GenBank
TM
accession number AF096871).
The deduced protein is 63% identical to the ER-type Ca
2⫹
-
ATPase reported from rice (5) (Fig. 1). The sequence identity
was 46% with SERCAs (sarco/endoplasmic reticulum calcium-
ATPases, see Ref. 28) and 56–64% with plant homologs from
tomato and Arabidopsis (7, 9). The sequence identity is greater
than 75% in the essential sequence motifs (see below). The
CAP1 protein includes all the domains highly conserved in
P-type Ca
2⫹
-ATPases. The 10 transmembrane domains pre-
dicted by hydropathy analysis are analogous to those in
SERCA-pumps (TM1 to TM10 in Fig. 1). A hydrophilic domain
between TM4 and TM5 of CAP1 contains a potential aspartyl
phosphorylation site within the CSDK motif and two ATP-
binding domains (Fig. 1) characteristic of all P-type ion pumps
(29). Six residues within TM4, -5, -6, and -8 required for high
affinity Ca
2⫹
transport (28) are all conserved in the maize clone
(Fig. 1). The transmembrane domains in CAP1 have different
degrees of identity to the animal SERCAs and the plant Ca
2⫹
-
pumps reported thus far. TM5 and TM6 of CAP1 are 80 and
88% identical to those of SERCAs, 92–96% with those in tomato
LCA1 and Arabidopsis ECA1 products, and 100% with those in
rice Os-Ca-ATPase. However, CAP1 differs in its sequence and
structure (⬃30% sequence identity) from the PM-type Ca
2⫹
-
pumps including those recently reported from cauliflower (8)
and Arabidopsis (6).
The sequence shown in Fig. 1 is compiled from the original
cDNA, CAP1 (which is short only by 19 amino acids from the
complete sequence), and an extension product of the 5⬘-se-
quence by rapid amplification of cDNA ends (RACE)-PCR re-
action. CAP1 is shorter at the N terminus by 25–32 residues
than its plant homologs from rice, tomato, and Arabidopsis, but
it shows a longer C-terminal tail (by ⬃40 residues) relative to
these SERCA-type pumps.
CAP1 Complements Yeast Mutants Defective in Ca
2⫹
-
pumps—The putative Ca
2⫹
-pumping function of CAP1 was
tested by expressing the clone in yeast mutants that lack the
Golgi-Ca
2⫹
-pump (pmr1) or both Golgi- and vacuole-located
Ca
2⫹
-ATPases as well as calcineurin (pmr1 pmc1 cnb1). As
shown in Fig. 2, Aand B, wild type yeast with functional
endogenous Ca
2⫹
-pumps grew on plates containing 10 mM
EGTA or MnCl
2
. However, mutants in the Golgi-located Ca
2⫹
-
ATPase (pmr1; Fig. 2, Aand B) or triple mutants lacking both
the endomembrane Ca
2⫹
-pumps (pmr1 pmc1 cnb1, also known
as K616; Fig. 2A, data not shown) failed to grow on EGTA or 3
mMMnCl
2
, as reported earlier (7, 30). CAP1 restored the
growth of both pmr1 and triple mutants on EGTA plates (Fig.
2A). The growth complementation was observed only in the
presence of galactose (Fig. 2A), the inducer of Gal1 promoter
under whose control the CAP1 cDNA was inserted. This sug-
gested that CAP1 was transcribed as well as translated, and
the product was assembled as a fully functional Ca
2⫹
-pumping
enzyme in yeast membranes. The transformants could not
grow on MnCl
2
(Fig. 2B, data not shown for K616), indicating
that CAP1 cannot transport Mn
2⫹
. Rabbit SERCA-pump was
also specific for Ca
2⫹
transport and failed to catalyze Mn
2⫹
transport (31). However, yeast PMR1 or Arabidopsis ECA1
products restored the growth of pmr1 on Mn
2⫹
(7, 32). The
specificity of CAP1 protein for Ca
2⫹
was further indicated by its
inability to restore the growth of the pmr2 mutant (deficient in
Na
⫹
/Li
⫹
efflux activity) on high lithium-containing medium
(Fig. 2C).
CAP1 Is Transcribed and Translated in the Yeast Mutant
K616 —We have determined whether the phenotypic comple-
mentation of yeast mutants by CAP1 was indeed due to the
expression of CAP1 cDNA in a galactose-dependent manner.
Total RNA preparations from K616 transformants grown in the
presence of glucose or galactose were probed with the 5⬘-end of
CAP1 cDNA. The results, presented in Fig. 3A, show that CAP1
transcripts are detectable only in pCAP1-transformed mutant
FIG.1.Amino acid sequence alignment of rice Os-Ca-ATPase
and the deduced CAP1 protein. The alignment was performed using
MacDNASIS software. Closed boxes indicate conserved amino acid res-
idues. Transmembrane regions are denoted by a line on the top of the
maize CAP1 and one below the Os-Ca-ATPase and numbered sequen-
tially from TM1 to TM10. The phosphorylation site (Asp
333
) and two
regions (483–488 and 681–696) that form the ATP-binding domain in
SERCA (29) are conserved in CAP1 (underlined in bold). Potential
Ca
2⫹
-binding sites (Glu
291
, Glu
759
, Asn
784
, Thr
787
, Asp
788
, and Glu
919
),
denoted by asterisks, within predicted transmembrane regions, TM4,
-5, -6, and -8 of CAP1 are required for Ca
2⫹
transport in the rabbit
SERCA.
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cells, and only when grown on galactose. The absence of CAP1
transcripts in glucose-grown cells is consistent with the repres-
sion of GAL1 promoter by glucose and the inability of glucose to
support the growth of the mutant in the absence of extracellu-
lar Ca
2⫹
(Fig. 2A). A strong inducibility of CAP1 in K616 cells
was also indicated by the detection of CAP1 signals using only
7.5
g of total RNA per lane.
We tested for the presence of CAP1 protein in the micro-
somes of transgenic yeast mutant. For this, monoclonal anti-
bodies were produced against a recombinant protein homolo-
gous to the central hydrophilic loop of CAP1 deduced amino
acid sequence (residues 342–576). This fragment was overex-
pressed in E. coli as a His-tagged protein; and the recombinant
protein was purified using a nickel column. The antisera raised
against CAP1 protein recognized an ⬃110-kDa protein in the
membranes isolated from K616 yeast cells transformed with
pCAP1 (Fig. 3B). The lower molecular mass band detected (⬃80
kDa) in the yeast membranes could be a proteolytic product of
the 110-kDa CAP1 product, as neither of the cross-reactive
bands was present in the membranes from the mutant trans-
formed with the vector alone (Fig. 3B).
The CAP1 Product Forms a Ca
2⫹
-dependent Phosphorylated
Intermediate Characteristic of P-type ATPases—We asked
whether the CAP1 product expressed in yeast mutants can
form a phosphorylated intermediate (E-P) characteristic of
Ca
2⫹
-ATPases, i.e. a hydroxylamine-sensitive and Ca
2⫹
-
dependent acyl-phosphate intermediate. As shown in Fig. 4,
the membranes from K616 cells expressing CAP1 showed one
32
P-labeled band (⬃110 kDa) in the presence of Ca
2⫹
. Further-
more, no signals were seen in membranes from yeast cells
transformed with vector alone (Fig. 4), indicating that the
⬃110-kDa phosphoprotein is most likely identical to the CAP1
product. The labeling of the 110-kDa band was dependent on a
short incubation time (⬃15 s, data not shown), which is con-
sistent with the rapid turnover rate of the phosphorylation
intermediate common for all P-type ATPases (33). Incubating
the reaction product with hydroxylamine completely removed
the label (Fig. 4), confirming that the phosphorylation was by
an acyl phosphate-linkage characteristic to P-type ATPases.
The phosphate labeling was dependent on the presence of Ca
2⫹
in the reaction mixture and enhanced by lanthanum (Fig. 4),
consistent with the slow turnover rate of the intermediate in
the presence of lanthanum. Thus, the E-P assay further con-
firmed that the CAP1 product may be a functional
Ca
2⫹
-ATPase.
CAP1 Expressed in Yeast Mutant Binds CaM-Sepharose in a
FIG.2. Complementation of yeast
mutants lacking either Golgi Ca
2ⴙ
-
pump (pmr1, AA542) or both Golgi
and vacuolar Ca
2ⴙ
-pumps (pmr1
pmc1; K616) by CAP1 expression. A,
CAP1 restores the growth of AA542 and
K616 yeast mutants on low Ca
2⫹
medium.
Wild type (W303), pmr1, and pmr1 pmc1
(K616) cells were transformed with the
vector (p426) alone or the chimeric plas-
mid containing the maize CAP1 clone
driven by Gal1 promoter. Cells were
streaked on SC-Ura plates containing 10
mMEGTA ⫹glucose or galactose and in-
cubated at 30
o
C for 4 days. B, CAP1
failed to restore the growth of yeast mu-
tants lacking Ca
2⫹
-pumps on Mn
2⫹
. Wild
type (W303) and pmr1 cells transformed
with p426 alone or the vector containing
the CAP1 were grown on SC-Ura plates
containing 3 mMMnCl
2
⫹glucose or ga-
lactose for 5 days at 30
o
C. C, CAP1 failed
to restore the growth of yeast pmr2 mu-
tant (defective in Na
⫹
/Li
⫹
efflux pump) on
high Li
⫹
-containing medium. Wild type
(W303) and pmr2 cells were transformed
with the vector alone or the chimeric plas-
mid containing the CAP1. Cells were
grown on SC-Ura plates containing glu-
cose or galactose supplemented with 150
mMLiCl for 5 days at 30
o
C.
FIG.3.CAP1 is transcribed and translated in the yeast mutant
K616. A, gel blot hybridization of yeast RNA with CAP1. 7.5
g of total
RNA from glucose (Glu)- or galactose (Gal)-grown K616 cells either
transformed with the vector alone (p426) or the vector containing CAP1
(pCAP1) is blotted after separation in a 1% agarose-formaldehyde gel
and probed with random-primed CAP1 and actin cDNA. B, protein gel
blot analysis of yeast microsomal proteins. Microsomes were prepared
from K616 cells transformed either with the vector alone (p426) or with
p426 containing the CAP1 clone (pCAP1) and probed with monoclonal
anti-24-kDa CAP1 protein antisera. 5
g of protein was loaded per lane.
Probing an identical blot with preimmune serum or anti-histidine an-
tisera did not give any signal. Size markers are shown on the left side
of the panel.
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Ca
2⫹
-dependent Manner—Since the CAP1 product appeared to
be divergent from the known ER-type Ca
2⫹
-ATPases by pos-
sessing an extended C-terminal tail rich in positively charged
residues interspersed by hydrophobic amino acids, it is possible
that the CAP1 product may be a CaM-regulated Ca
2⫹
-ATPase.
The CaM binding nature of the CAP1 protein expressed in the
yeast mutant K616 was assessed using CaM-affinity chroma-
tography. Detergent-solubilized microsomal proteins (0.75–1
mg) were loaded on a 2-ml column of CaM-Sepharose in the
presence of Ca
2⫹
. The column was washed with excess column
buffer and eluted with 10 mMEGTA. The second and third
column fractions of the eluent showed enrichment of poly-
petides at 100-, 60-, 40-, and 33-kDa region as detected by
silver staining (data not shown). The flow-through (40
gof
protein) and the eluent (3–5
g protein) were probed by anti-
CAP1 antisera. As shown in Fig. 5, a cross-reactive band at
105–110 kDa was apparent only in the eluent and none in the
flow-through. The results indicate that CAP1 product ex-
pressed in K616 is capable of interacting with calmodulin and
is most likely regulated by CaM in vivo.
Microsomes from the CAP1-expressing Yeast Mutant Mediate
Calmodulin-stimulated Ca
2⫹
Transport—To determine further
whether the CAP1 complementation of yeast mutants defective
in Ca
2⫹
-pumps was indeed resulted by the restoration of cal-
cium transport, we have tested the Ca
2⫹
transport activity of
microsomes isolated from the triple mutant K616 expressing
CAP1. The mutant transformed with vector alone showed back-
ground Ca
2⫹
uptake (40–55% of the CAP1 transformed cells).
This is apparently driven by H
⫹
/Ca
2⫹
antiporter as indicated
by its abolition by gramicidin (24, 34) or nitrate (data not
shown). The addition of bafilomycin (24, 34) was not tested, as
gramicidin alone was effective in eliminating the antiporter-
driven Ca
2⫹
transport in the mutant at the pH/ionic conditions
of our assays. Gramicidin was routinely added to our assays to
eliminate interference from the antiporter activity. The CAP1-
mediated transport activity was dependent on the presence of
ATP in the assay buffer and was abolished by heating the
membranes at 90 °C for 3 min (data not shown). The Ca
2⫹
uptake increased up to 5 min (Fig. 6A) and reached a plateau
within 10 min (data not shown). Addition of the ionophore
A23187 released most of the membrane-accumulated
45
Ca
2⫹
,
indicating that the transport was against a concentration gra-
dient (Fig. 6A). Ca
2⫹
dependence assay of the transport indi-
cated that the Ca
2⫹
uptake by CAP1 saturates at ⬃5
Mfree
Ca
2⫹
in the medium (Fig. 6B).
The CaM regulation of CAP1 was further confirmed by test-
ing the effect of exogenous CaM or W7, a CaM inhibitor on the
CAP1-restored Ca
2⫹
transport activity of the triple mutant.
The results, presented in Fig. 6C, show up to a 2-fold stimula-
tion of Ca
2⫹
transport activity by an external addition of CaM,
indicating that the CAP1 activity may be calmodulin-regu-
lated. The calmodulin inhibitor, W7, decreased the
45
Ca trans-
port by 50% even in the absence of supplemental calmodulin
(data not shown) indicating that CAP1 product may be associ-
ated with endogenous CaM. With supplemental CaM in the
assay buffer, W7 caused a very high background retention of
45
Ca
2⫹
on the filters. Therefore, its effect on the Ca
2⫹
transport
activity of the microsomes in the presence of CaM could not be
reliably ascertained (data not shown). In summary, these re-
sults provide further evidence that CAP1 is a calmodulin-reg-
ulated Ca
2⫹
-ATPase.
Peptides from CAP1 C Terminus Bind Ca
2⫹
/Calmodulin—
Given the CaM binding nature of recombinant CAP1 and CaM
stimulation of its activity, we expected to find a potential CaM-
binding domain in its sequence. Taking analogy to the animal
calmodulin-regulated pumps and the presence of an extended C
terminus into consideration, we searched for a putative CaM-
binding domain at the C terminus of CAP1. Although not
conserved in their primary sequence, CaM-binding domains
most commonly form basic amphipathic
␣
-helices (35). The C
terminus of CAP1 showed helical structures with clusters of
positively charged amino acids. Three overlapping regions (be-
tween 995 and 1049 residues, Fig. 7A) were selected based on
the properties of their helical wheel projections. Synthetic pep-
tides were made corresponding to these sequences and tested
for their ability to shift the mobility of calmodulin (recombinant
or purified from roots) in native acrylamide gels (19). Of the
three peptides tested, only the two distal peptides (peptides P2
and P3, Fig. 7A) with an overlapping sequence of KQKASSER-
RLTFD bound CaM and shifted its mobility in a Ca
2⫹
-depend-
ent manner (Fig. 7, B–D). Between P2 and P3, the latter caused
a greater retardation in the CaM mobility in the presence of
urea (Fig. 7B). However, electrophoresis of CaM䡠peptide com-
plexes in the absence of urea led to a complete shift of CaM by
P2 at equimolar concentrations (Fig. 7C). The two peptides (P2
and P3) that caused a mobility shift were highly basic in their
FIG.4.The CAP1 product from yeast microsomes forms a Ca
2ⴙ
-
dependent phosphorylated intermediate characteristic of
P-type ATPases. Microsomes were prepared from K616 cells trans-
formed either with the vector alone (p426) or with p426 containing the
CAP1 clone (pCAP1) and used for the phosphoenzyme assay (5–7.5
g
of protein per lane). Microsomes were incubated with [
␥
-
32
P]ATP in the
presence of CaCl
2
(Ca), EGTA, or LaCl
3
(La) as described under “Ex-
perimental Procedures.” In the lane labeled NH
2
OH, the trichloroacetic
acid-precipitated proteins were solubilized in the presence of hydoxy-
lamine. Size markers are shown on the left side of the figure.
FIG.5.CAP1 expressed in yeast mutant binds CaM-Sepharose
in a
2ⴙ
-dependent manner. Protein gel blot analysis of root microso-
mal CaM-binding proteins. Monoclonal antisera raised against the pu-
rified 24-kDa CAP1 product were used to probe a gel blot of the CaM-
binding fraction of maize root microsomal proteins (5
g). Probing an
identical blot with preimmune serum or anti-histidine antisera did not
give any signal.
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overall charge (pI ⫽12) in contrast to the P1 peptide (pI ⫽6.36)
that failed to induce a shift. However, P
2
and P
3
did not bind to
other acidic proteins such as soybean trypsin inhibitor or bo-
vine serum albumin (data not shown), under identical condi-
tions. This indicated that the two CAP1 peptides bound cal-
modulin by specific hydrophobic interactions and not due to
electrostatic attraction.
The binding of CaM to its targets induces structural changes
in CaM as well as the target peptides/proteins. Binding is
accomplished by a change in conformation of the central
␣
-helix
of CaM to a random coil allowing the globular lobes of CaM to
engulf the target peptide (36). In turn, the target peptides may
attain a greater helicity upon CaM binding. Hence, spectropo-
larimetry has been a useful tool to follow the helicity changes in
CaM䡠target complexes (19). CAP1 peptide and CaM interac-
tions were monitored using CD spectra. Calmodulin displayed
a CD spectrum typical of helical proteins with minima at 222
and 208 nm and a maximum at 190 nm (data not shown). The
peptides themselves showed random conformation as indicated
by the spectra of uncomplexed peptides (data not shown). Fig.
8Apresents the difference spectra resulting after the subtrac-
tion of the spectrum of individual peptide䡠CaM complex from
that of CaM alone. When co-incubated with calmodulin, pep-
tides P2 and P3 induced a change in the molar ellipticity of
CaM at 208 and 222 nm, whereas P1 induced nonspecific
changes (Fig. 8A). The changes induced by P2 and P3 indicate
a decreased helicity of CaM and an increase in the helicity of
the two peptides (19). Furthermore, the interaction was de-
pendent on Ca
2⫹
, as these conformational changes were abol-
ished by EGTA (Fig. 8B). The absence of a helicity increase in
P1 confirmed that CAP1 residues included in this peptide are
not involved in the interaction of the pump with CaM, in
accordance with our results from CaM-gel shift assays (data
not shown). Furthermore, CD spectral analysis also showed
that P3 caused greater decrease in the helicity of CaM than P2
did (Fig. 8B), confirming that in addition to KQKASSER-
RLTFD other residues in the P3 peptide are important for the
interaction of the pump with calmodulin. A recombinant pep-
tide homologous to the C terminus that encompasses the CaM-
binding peptides was expressed in E. coli. This longer peptide
can be purified to homogeneity by CaM-affinity chromatogra-
phy and interacts with CaM in filter binding assays (data not
shown). Since this peptide is toxic to E. coli and could not be
overexpressed, synthetic peptides will be used to fine map the
CaM-binding domain.
Antibodies Against a Recombinant CAP1 Polypeptide Recog-
nize a Single Maize Microsomal Protein Purified by CaM-Affin-
ity Chromatography—We attempted to identify and character-
ize the CAP1 cognate protein from maize root tissue, using
CAP1 antisera. Although most of the monoclonal antisera gave
a reactivity at high titer with the recombinant polypeptide
expressed in E. coli (⬎16,000-fold dilution) or yeast (1:4000),
none of the clones recognized any polypeptide in total maize
microsomes or after fractionation on sucrose gradients (data
not shown). This indicated that the cognate protein, if present,
is in very low abundance in maize tissues. Since our results
with the CAP1 product suggested that it is a CaM-binding
protein (Figs. 5–8), we enriched maize root microsomal pro-
teins on CaM-affinity chromatography and then probed with
CAP1 antisera. Microsomal CaM-binding preparations showed
a major polypeptide of ⬃110 kDa and 5–6 additional bands of
varying sizes (Fig. 9A). The CAP1 antibodies cross-reacted only
with the 110-kDa polypeptide (Fig. 9B), indicating that the
CAP1 cDNA encodes a single microsomal protein that binds
CaM by itself or through interaction with a calmodulin-binding
protein.
A 110-kDa Protein from the CaM-Sepharose Binding Frac-
tion of Maize Microsomes Directly Interacts with Ca
2⫹
/Cal-
modulin—The ability of putative CAP1 cognate protein to in-
FIG.6. Characterization of Ca
2ⴙ
transport activity driven by
CAP1 in yeast microsomes. A, yeast microsomes from vector or CAP1-
transformed cells were isolated and assayed for ATP-dependent Ca
2⫹
transport activity as described under “Experimental Procedures.” At the
end of 5 min, 1
Mof the ionophore A23187 was added in a replicate assay.
Vector-transformed cells showed no ATP-stimulated activity in the pres-
ence of gramicidin (routinely added at 50
Mto the assay medium). The
calculated free Ca
2⫹
in this assay was 57 nM. However, the linearity of
transport was maintained only for the first 5 min, even at higher free
Ca
2⫹
concentrations tested (1 or 5
M). B, Ca
2⫹
dependence of
45
Ca
transport driven by CAP1. CaCl
2
/EGTA mixtures were used to buffer free
Ca
2⫹
concentration as described under “Experimental Procedures.” C,
effect of supplemental calmodulin was tested using recombinant maize
calmodulin. Bovine serum albumin was substituted for CaM to provide
equimolar concentration of protein in all the tubes. Background activity in
the absence of ATP was subtracted from each corresponding assay with
ATP. Values are means of duplicate samples from a single assay and
represent results of three separate membrane preparations.
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teract independently with CaM was tested by the overlay of
HRP-labeled CaM on gel blots of microsomal CaM-Sepharose
eluents. With brief exposures to x-ray film, only a 110-kDa
band was found to bind calmodulin in these assays (Fig. 10A),
although longer exposures revealed additional CaM-binding
proteins in the preparation (data not shown). The binding was
abolished when CaCl
2
was replaced by EGTA from the binding
buffer (data not shown). Calcineurin was used as a positive
control in the assay, and only the 58.6-kDa large subunit
(which is known to be the CaM-binding subunit of the holopro-
tein) was bound by calmodulin (Fig. 10A). Taken together,
CaM-affinity chromatography, CaM overlay assay, and immu-
noblotting experiments reveal that the CAP1 cognate protein
in maize tissues is a calmodulin-binding protein.
The 110-kDa polypeptide migrated faster in the presence of
Ca
2⫹
-chelator EGTA in acrylamide gels, i.e. asa⬃100-kDa
polypeptide (Fig. 10B). This mobility shift, which is character-
istic of many Ca
2⫹
-binding proteins (e.g. Refs. 37 and 38 and
references therein), indicated that the CAP1 cognate polypep-
tide binds Ca
2⫹
, in addition to calmodulin. This is in accord-
ance with the putative function of the CAP1 product as a
Ca
2⫹
-pumping enzyme and conservation of the putative Ca
2⫹
-
binding residues (Glu
291
, Glu
759
, Asn
784
, Thr
787
, Asp
788
, and
Glu
919
) within the predicted transmembrane regions, TM4, -5,
-6, and -8 (Fig. 1).
The CaM-Sepharose Fraction Has a 110-kDa Polypeptide
That Forms a Ca
2⫹
-dependent Phosphorylated Intermedi-
ate—We also investigated if the 110-kDa CaM-binding protein
of maize microsomes can form Ca
2⫹
-dependent acyl-phosphate
intermediate. As shown in Fig. 10C, the CaM-binding prepa-
ration showed one
32
P-labeled band in the presence of Ca
2⫹
.
The phosphoprotein was similar in its molecular size (⬃110
kDa) to that of the anti-CAP1 cross-reactive polypeptide. The
labeling of the polypeptide was also dependent on a short
incubation time, sensitive to hydroxylamine dependent on the
presence of Ca
2⫹
in the reaction mixture and enhanced in the
presence of lanthanum (Fig. 10C). These results suggest that
the putative CAP1 cognate protein in maize membrane may be
a functional Ca
2⫹
-ATPase.
FIG.8.Circular dichroism spectra of calmodulin and its com-
plexes with CAP1 peptides. A, difference spectra obtained by sub-
tracting the spectrum of 3
Meach peptide ⫹3
MCaM from that of 3
MCaM alone; symbols are as follows: circles, P1; squares, P2; dia-
monds, P3. The binding buffer contained 0.5 mMCaCl
2
. The mean
residue ellipticity is expressed in deg䡠cm
2
/dmol of amino acid residues in
the peptide. B, Ca
2⫹
dependence of helicity changes in the CaM䡠peptide
complexes. The difference spectrum for P3 peptide, indicated by circles,
was obtained in the presence of CaCl
2
(as described in A) and the one
indicated by squares was obtained in the absence of CaCl
2
i.e. in the
presence of 2 mMEGTA.
FIG.7.Peptides from the C terminus of CAP1 bind to calmodulin. A, synthetic peptides (P1, P2, and P3) identical in their sequence to the
C terminus of deduced CAP1 were used to test the affinity of CAP1 product to calmodulin. The location of each peptide in the CAP1 sequence is
shown by the number of start and end residues. The sequence overlap within the peptides is indicated by an underline. B, interaction of CAP1
peptides with calmodulin as studied by native urea-acrylamide gel electrophoresis. 150, 300, 750, and 1500 pmol of P1 (lanes 2–5), P2 (lanes 6–9),
or P3 (lanes 10 –13) were incubated with 300 pmol of calmodulin in the presence of 4 Murea and 100
MCaCl
2
for 1 h. The peptide-protein
complexes were analyzed in a native 4 Murea, 12.5% acrylamide gel as described by Erickson-Viitanen and DeGrado (19). Lanes 1 and 14 show
the migration of free CaM. Electrophoresis of free peptides in the same gel did not result in any bands. C, interaction of CAP1 peptides with
calmodulin as studied in native gels lacking urea. 150 and 300 pmol of P1 (lanes 2 and 3), P2 (lanes 4 and 5), or P3 (lanes 6 and 7) were incubated
with 300 pmol of calmodulin in the presence of 100
MCaCl
2
for 1 h. The peptide-protein complexes were analyzed in native acrylamide gels (19).
Lane 1 shows the migration of free CaM. D, Ca
2⫹
dependence of the CaM interaction with CAP1 peptides. The protein and peptide incubations were
as in lanes 1–5 of B, except that Ca
2⫹
was replaced by 2 mMEGTA in the binding and gel running buffers (to lower the free Ca
2⫹
concentration
to near zero). Results are shown for P2 and were similar with P1 and P3 peptides. Lane 1 is free CaM and lanes 2–5 are 150, 300, 750, and 1500
pmol of peptide incubated with 300 pmol of CaM.
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CAP1 Transcripts Are Low Abundant and Induced by Anoxia
in Maize Roots—Our interest is to elucidate the role of this
chimeric Ca
2⫹
-pump in intracellular signaling, particularly in
the Ca
2⫹
-mediated signaling of anoxia. As a first step toward
this goal, we investigated the abundance and induction pat-
terns of CAP1 message in maize roots by different environmen-
tal stresses. Gel blot hybridization and RT-PCR experiments
indicated that that CAP1 transcripts are of very low abundance
in maize tissues, as was the concentration of its cognate protein
in the microsomes. Consistent detection of signals in the RNA
gel blots required the purification of poly(A) RNA. Specific
hybridization signals could often be detected by loading ⬎40
g
of total RNA per lane (data not shown). There was only single
transcript class of about 6 kilobase pairs long indicating that
there could be a processing regulation of the primary tran-
script. Despite the low abundance of its transcripts in maize
roots, the CAP1 cDNA was isolated by screening ⬍2⫻10
5
plaques from 6-h anoxic maize root cDNA library. This indi-
cated that the CAP1 transcript abundance might increase dur-
ing anoxia. We tested the induction of CAP1 message under
anoxia as well as under other abiotic stresses. The CAP1 tran-
scripts were induced and maintained at 2–3-fold greater than
aerobic levels for 2–4 h of anoxia (Fig. 11A). Furthermore, the
faster migration of CAP1 transcripts at4hofanoxia is also
suggestive of a potential processing regulation of the primary
transcript. Heat shock also caused a mild induction of CAP1,
but salt, cold, and osmotic treatments were ineffective (Fig.
11A).
CAP1 Appears to Be Encoded by a Single Copy Gene in Maize
Genome—Genomic DNA gel blot hybridization was done to
investigate if there is a gene family encoding CAP1 homologs in
the maize genome. Probes from three different regions of CAP1
clone (including the central hydrophilic loop) were used for
hybridization at medium or low stringency. Results indicate
that there are only one or two genes that encode CAP1 in the
maize genome (Fig. 11B). This prediction is in agreement with
the protein gel blot analysis (Fig. 9). The Southern hybridiza-
tion results also suggested that there is restriction fragment
length polymorphism among the genotypes tested (Fig. 11B).
DISCUSSION
In mammalian cells, molecular and biochemical evidence
shows that the CaM-regulated Ca
2⫹
-pump is located on the
plasma membrane and is divergent in its sequence and struc-
tural features from the SERCA-type Ca
2⫹
-pumps. Evidence
has accumulated that there are calmodulin-regulated Ca
2⫹
-
ATPase activities in plant cells (39). However, unlike in animal
cells, such activities have been reported both from purified
plasma membrane preparations (40–42) as well as enriched
endomembranes (e.g. Refs. 13, 22, and 42–44). The presence of
multiple calmodulin-regulated Ca
2⫹
-pumping activities and
their distribution to different cellular compartments indicate a
crucial role for these enzymes in plant cell signaling. Isolation
and functional characterization of genes encoding these multi-
ple Ca
2⫹
-pumps would facilitate the unraveling of mechanisms
of cellular Ca
2⫹
homeostasis and attendant pathways of cellu-
lar communication. Recently, Malmstrom et al. (8) and Harper
et al. (6) have cloned cDNAs for CaM-regulated Ca
2⫹
-pumps
from cauliflower and Arabidopsis (BCA1 and ACA2, respective-
ly). These clones are related to the mammalian plasma mem-
brane Ca
2⫹
-ATPases, although the CaM-binding domain in the
plant clones is located at the N terminus. CAP1 presents yet
another novel type of CaM-binding Ca
2⫹
-pump in plants. Se-
quence homology to SERCAs, growth and functional comple-
mentation of yeast mutants deficient in Ca
2⫹
-ATPases, binding
affinity of synthetic peptides and transgenic CAP1 protein to
calmodulin, CaM-stimulated Ca
2⫹
transport activity in yeast
microsomes, antigenic identity of CAP1 product with a Ca
2⫹
-
and CaM-binding microsomal protein from maize roots, prop-
erties of the phosphoenzyme formed by cognate proteins, pro-
vide strong support to our proposal that CAP1 encodes a calm-
odulin-binding Ca
2⫹
-ATPase in maize. It differs from the
canonical mammalian CaM-regulated Ca
2⫹
-pumps in that it
has greater overall sequence identity with the SERCA-type
Ca
2⫹
-ATPases (e.g. Refs. 7, 29, and 45). At the same time, it is
similar to the animal PM-type pumps in having a CaM-binding
domain in the C-terminal tail. Thus, CAP1 shares features of
both the PM- and ER-type Ca
2⫹
-pumps of mammalian systems.
However, it is very divergent from BCA1 or ACA2 (the plant
homologs of PM-type pumps) in the overall sequence as well as
in the location of its CaM-binding domain. Thus, the presence
of multiple CaM-regulated activities distributed on more than
one cellular membrane and novel type of genes encoding these
activities indicate that plant signaling pathways or compo-
nents involved may not always fit the animal paradigm.
Previously, evidence was presented for the presence of CaM-
regulated Ca
2⫹
-ATPases in young maize seedling shoots (11,
13, 14). However, the polypeptides that were attributed to
belong to Ca
2⫹
-pump were of two different size ranges. Briars
et al. (11) purified an enzyme on CaM-affinity columns, and
this preparation showed a 140-kDa polypeptide that cross-
reacted with antisera for the mammalian CaM-binding Ca
2⫹
-
ATPase. Later, this polypeptide was confirmed to be a Ca
2⫹
-
pump by reconstitution studies as well as phosphoenzyme
analysis (12, 13). On the other hand, Logan and Venis (14)
identified a 102-kDa polypeptide of maize microsomes as CaM-
binding Ca
2⫹
-ATPase based on its cross-reactivity with anti-
SERCA antisera as well as binding to CaM. However, no fur-
ther studies have been reported on this protein. The product of
CAP1 clone has a predicted mass of 113 kDa, which is also in
the size range of the polypeptide recognized by CAP1 antisera
in maize. This is much smaller than the 140-kDa Ca
2⫹
-pump
purified by Theodoulou et al. (13). The cross-reactivity of the
140-kDa protein with the antisera for mammalian PM Ca
2⫹
-
pump further indicates that these two pumps are most likely
divergent even in their sequence. In contrast, the 102-kDa
FIG.9. Antibodies to CAP1 24-kDa polypeptide recognize a
single polypeptide in the calmodulin-binding proteins of maize
root microsomes. A, SDS-polyacrylamide gel electrophoresis analysis
of root microsomal calmodulin-binding proteins. Microsomal proteins
from 3-day-old maize seedling roots were fractionated on CaM-Sepha-
rose column, and the bound proteins (5
g) were resolved in a 6–12%
gradient acrylamide gel. The proteins were stained in silver. B, protein
gel blot analysis of root microsomal CaM-binding proteins. Monoclonal
antisera raised against the purified 24-kDa CAP1 product were used to
probe a gel blot of the CaM-binding fraction of maize root microsomal
proteins (5
g). Probing an identical blot with preimmune serum or
anti-histidine antisera did not give any signal. Size markers are shown
on the left side of the figures.
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CaM-binding putative Ca
2⫹
-ATPase reported by Logan and
Venis (14) is in the size range of the CAP1 product. Further-
more, these authors showed that a similar polypeptide cross-
reacted with antibodies for a region conserved in all SERCA-
type-pumps, including the CAP1 (Refs. 5, 7, 9, and 46; Fig. 1).
Our studies further indicate that the CAP1 protein possesses
its CaM-binding domain at the C terminus, and the evidence is
3-fold. CaM-affinity chromatography indicated that a recombi-
nant polypeptide corresponding to the last 100 residues of the
C terminus bound calmodulin in the presence of Ca
2⫹
(data not
shown). CaM mobility shift assays demonstrated that shorter
peptides within this C-terminal tail bound to CaM in calcium-
dependent manner and retarded its mobility in acrylamide-
urea gels (Fig. 7). Furthermore, co-incubation of CaM with
CAP1 peptides resulted in characteristic conformational
changes typical to CaM䡠target complexes, as revealed by the
CD spectra (Fig. 8). These studies were also indicative of the
amino acid residues involved in the interaction of CAP1 with
CaM. Of the three peptides tested, only P2 and P3 were effec-
tive in binding to CaM in gel shift as well as spectropolarimet-
ric assays. The overlapping sequence KQKASSERRLTFD ap-
pears to be critical for CaM binding, although CaM gel
FIG. 10. Calmodulin and Ca
2ⴙ
binding activity of maize root microsomal proteins fractionated on CaM-Sepharose. A, the CaM-
binding fraction (5
g) of maize root microsomes was resolved in a denaturing 6–12% gradient acrylamide gel (lane 1), blotted onto nitrocellulose,
and tested for CaM-binding proteins in CaM overlay assays. The preparation of HRP-labeled CaM is described under “Experimental Procedures.”
Calcineurin (1–2
g, Sigma) was loaded in lane 2 as a positive control. The HRP signal was detected by enhanced chemiluminescence. B,
Ca
2⫹
-dependent mobility shift of 110-kDa polypeptide. The CaM-binding fraction was analyzed in a denaturing 7–15% gradient acrylamide gel in
the presence of 0.5 mMCaCl
2
(⫹)or2mMEGTA (⫺). C, phosphoenzyme analysis of maize microsomal CaM-binding proteins. CaM-binding proteins
(5
g) from maize microsomes were used for phosphorylated intermediate analysis as described in Fig. 4. Size markers are shown on the left side
of the figures.
FIG. 11. CAP1 is expressed in low abundance in maize roots and may be encoded by a single gene. A, RNA gel blot analysis of CAP1
transcripts. Maize seedlings were exposed to none (C), 2, 4, and 24 h of submergence (anoxia),1hoftemperature shock at 10 or40°C,5hofsalt
stress with 0.1 MNaCl (SS), or osmotic shock (OS) with 0.1% polyethylene glycol 8000 for 4 h. Poly(A) RNA was purified from the root tissue of
treated seedlings and resolved in formaldehyde-agarose gels (2
g per lane). The gel was blotted and probed with
32
P-labeled CAP1 cDNA. The blot
was subsequently hybridized to a constitutive probe, 1055, and CAP1 signals were normalized as described earlier (2). The normalized intensities
of CAP1 are shown in the grid at the bottom. B, genomic DNA gel blot analysis of CAP1. 20
g of genomic DNA from the maize inbred B73 or Mo17
was digested with EcoRI (E), HindIII (H), or HpaI(Hp), fractionated in a 0.8% agarose gel and blotted onto nylon membrane. The blot was probed
with a fragment of CAP1 that encodes the central hydrophilic loop. Probes from 5⬘- and 3⬘-ends of CAP1 hybridized to single bands of different sizes
in the two genotypes.
Maize cap1 Encodes a Novel SERCA-type Calcium-ATPase21686
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retardation assays and CD spectra indicate additional residues
in the extreme C terminus may enhance the interaction.
A high degree of sequence similarity between CAP1 and
SERCAs indicates that the CAP1 protein may be located on the
ER membranes. However, the tomato LCA1 product, despite its
similarity to the mammalian ER pumps, is not ER-localized but
is distributed on the tonoplast and the plasma membrane (45).
This has also been the case with the cauliflower calmodulin-
regulated BCA1 product, which is a PM-type pump (42). It is
not known if the CAP1 product is also localized on more than
one membrane in maize cells. Nevertheless, the products of
ECA1 and ACA2 from Arabidopsis are confined to the ER/
intracellular membranes. In fact, biochemical evidence sug-
gests that the CaM-regulated Ca
2⫹
pumping activity in maize
seedlings is predominantly distributed on the endomem-
branes, either the ER or the tonoplast (Refs. 13, 14, 47, and 48;
but also see Ref. 40).
The low abundance of the CAP1 protein and transcripts in
maize tissues indicates a tight regulation of CAP1 expression.
Furthermore, regulation of the Ca
2⫹
transport activity by cal-
modulin suggests the involvement of CAP1 product in a feed-
back attenuation of cytosolic Ca
2⫹
concentration during cell
stimulation. A stringent regulation of Ca
2⫹
sequestration from
the cytosol should allow the Ca
2⫹
-dependent signaling pro-
cesses to continue without the cell attaining cytotoxic levels of
free Ca
2⫹
. Induction of CAP1 transcripts in maize roots only
during the early hours of anoxia indicates such a regulation of
Ca
2⫹
homeostasis in the O
2
-deprived maize cells.
Acknowledgments—We thank Alan Bennett (University of Califor-
nia, Davis) for providing the tomato LCA1 cDNA clone; Kyle Cunning-
ham (The Johns Hopkins University, Baltimore) for the gift of yeast
strains; Don Briskin (University of Illinois, Urbana) for help with Ca
2⫹
transport assays; Raymond Zielinski (University of Illinois, Urbana) for
suggestions on CaM gel shift assays; and Daniel Bush (University of
Illinois, Urbana), as well as Douglas Bush (University of California,
Santa Barbara), for helpful comments on the manuscript. We are also
thankful to our undergraduate students Graham Englund, Shun Pa,
and Andrew Miller for their enthusiasm and excellent laboratory
assistance.
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Maize cap1 Encodes a Novel SERCA-type Calcium-ATPase 21687
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Chalivendra C. Subbaiah and Martin M. Sachs
Calmodulin-binding Domain
Calcium-ATPase with a
Encodes a Novel SERCA-typecap1Maize
FUNCTION AND BIOGENESIS:
MEMBRANE TRANSPORT STRUCTURE
doi: 10.1074/jbc.M001484200 originally published online April 17, 2000
2000, 275:21678-21687.J. Biol. Chem.
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