From the United States Department of
Agriculture/Agricultural Research Service Children's Nutrition
Research Center and the § Department of Human and Molecular
Genetics, Baylor College of Medicine, Houston, Texas 77030 and the
¶ Vegetable and Fruit Improvement Center, Texas A&M University,
College Station, Texas 77845
Received for publication, October 23, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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Regulation of Ca2+ transporters
is a vital component of signaling. The Arabidopsis
H+/Ca2+ exchanger CAX1 contains an
N-terminal autoinhibitory domain that prevents Ca2+
transport when CAX1 is heterologously expressed in yeast. Using a yeast
screen, we have identified three different proteins that activate CAX1.
One of these, CXIP1
(CAX-interacting
protein-1; 19.3 kDa) has amino acid similarity
to the C terminus of PICOT (protein kinase
C-interacting cousin of
thioredoxin) proteins. Although PICOT proteins are found in
a variety of organisms, a function has not been previously ascribed to
a plant PICOT protein. We demonstrate that CXIP1 activated the CAX1
homolog CAX4, but not CAX2 or CAX3. An Arabidopsis homolog
of CXIP1 (CXIP2) weakly activated CAX4, but not CAX1. In a yeast
two-hybrid assay, CXIP1 interacted with the N terminus of CAX1. In
competition analysis, CXIP1 and a CAX1 N-terminal peptide appeared to
bind to similar N-terminal domains of CAX1. Chimeric CAX3 constructs
containing the N terminus of CAX1 were activated by CXIP1. In
Arabidopsis, CXIP1 transcripts, like
CAX1, accumulated in response to different metal
conditions. This work thus characterizes a new class of signaling
molecules in plants that may regulate CAX transporters in
vivo.
Ca2+ signal transduction requires the judicious
control of cytosolic Ca2+ levels. Cytosolic
"Ca2+ spikes" are either directly or indirectly
translated into biological responses that govern all aspects of growth
and development (1, 2). Endomembrane Ca2+ transporters are
believed to play an important role in specifying the duration and
amplitude of these cytosolic Ca2+ fluctuations (2, 3).
Understanding the regulatory mechanisms of these Ca2+
transporters is a fundamental component in dissecting signaling specificity.
Expression in yeast has facilitated the cloning of numerous plant
genes, including plant endomembrane Ca2+ transporters (4,
5). Initially, plant H+/Ca2+ antiporter genes
were cloned by their ability to suppress the Ca2+-hypersensitive phenotype of a Saccharomyces
cerevisiae mutant (4, 6). These genes are termed
CAX for cation exchangers, and CAX1 from Arabidopsis thaliana is a high capacity
Ca2+ transporter. Arabidopsis appears to have up
to 11 other putative cation/H+ antiporters (CAX2-11 and
MHX) (7). CAX1, CAX2, CAX4, and MHX have been shown to localize to the
plant vacuole (8-11). The activity of CAX1 appears to be regulated by
an N-terminal autoinhibitory domain that was absent in the initial
clone characterized by heterologous expression in yeast (12, 13).
Ectopic expression of deregulated CAX1 missing the N-terminal
autoinhibitor (sCAX1) in tobacco increases Ca2+ levels in
the plants and causes numerous stress-sensitive phenotypes often
associated with Ca2+ deficiencies (14, 15). Thus, a wide
range of environmental responses appear to require regulation of CAX1
transport activity; however, the mechanism by which CAX1 transport
becomes activated is currently not understood.
From recent studies, it has become clear that the N termini of
particular plant Ca2+-ATPases act as points of convergence
between Ca2+-signaling molecules that can both positively
and negatively regulate Ca2+ transport (3, 5). A
Ca2+-dependent protein kinase competes with
calmodulin for binding to the N termini of these
Ca2+-ATPases to negatively regulate pump activity (16).
Interestingly, the N terminus of CAX1 does not contain a
calmodulin-binding site, and biochemical studies show that CAX1 cannot
be activated by exogenous calmodulin (12). Moreover, expression of an
activated Ca2+-dependent protein kinase in
yeast strains harboring CAX1 does not alter
activity.1 These findings
strongly suggest that a unique set of signal transduction molecules
physically interact with the N terminus of CAX1 to modulate Ca2+ transport.
We propose that one possible mechanism of activation of CAX1 is through
a protein cofactor that directly interacts with the antiporter. In this
work, we have utilized autoinhibited CAX1 in a yeast screen to identify
and characterize plant gene products that activate CAX1-mediated
H+/Ca2+ transport. We have characterized the
specificity of this activation and the regions of CAX1 involved in the
physical interaction between CAX1 and one of these activators. This
study details a new class of signaling molecules in plants that may be
involved in activation of H+/Ca2+ transporters
in plants.
Yeast Strains and Plant Materials--
S. cerevisiae
strain K667 (MATa
cnb1::LEU1
pmc1::TRP1 vcx1 Yeast Transformation and Screening of a cDNA
Library--
Yeast strain K667 expressing CAX1, which is
hypersensitive to high concentrations of Ca2+, was
transformed with an A. thaliana (ecotype Landsberg) cDNA library constructed in the episomal yeast-Escherichia coli
shuttle vector pFL61 (19). Stable transformants were selected on
synthetic complete medium lacking His and Ura (20). Of these,
Ca2+ Tolerance Assay--
Yeast cells expressing a
CAX gene (CAX1, CAX3, or
CAX4 or chimeric CAX constructs) with the CXIP1
gene were assayed on YPD medium supplemented with 200 mM
CaCl2 (13, 21).
DNA Constructs and Site-directed Mutagenesis--
Chimeric CAX
constructs sCAX3- RNA Gel Blotting Analysis--
A. thaliana (ecotype
Columbia) seeds were germinated in artificial soil (Metro-Mix 200, Scotts, Marysville, OH) and grown under continuous light. Sample
preparation and RNA extraction were performed as previously described
(14). To analyze gene expression, 20 µg of total RNA was loaded onto
a formaldehyde-containing 1.0% agarose gel and blotted onto nylon
membrane (Amersham Biosciences, Buckinghamshire, United Kingdom).
Filters were hybridized overnight at 42 °C with a
32P-labeled gene-specific probe in 2× SSC and 50%
formamide, washed twice with 2× SSC and 0.1% SDS and twice with 0.1×
SSC and 0.1% SDS (both at 55 °C), and exposed to x-ray film
(Eastman Kodak Co.) at Preparation of Microsomal Membrane-enriched Vesicles and
Ca2+ Transport Assay--
Microsomal membrane-enriched
vesicles were isolated from yeast as described previously (13).
Time-dependent H+/Ca2+ transport
into endomembrane vesicles was examined using the direct filtration
method as described previously (12). Measurement of Ca2+
transport in the presence of a synthetic peptide was performed as
previously described (13).
Yeast Two-hybrid Interaction Assay--
The yeast two-hybrid
system used in this study was described previously (13, 18). The two
CAX1 N-terminal fragments (residues 1-65 and 37-73) were amplified by
PCR and cloned in-frame into the NcoI and BamHI
sites of pAS2. CXIP1 was amplified by PCR and cloned in-frame into the
NcoI and XhoI sites of pACT2. Protein expression
in yeast was confirmed as previously described (13).
Cloning and Sequencing of Arabidopsis cDNAs That Activate
CAX1--
A pmc1vcx1cnb1 yeast strain (K667) lacking the
vacuolar Ca2+ transporters Pmc1 and Vcx1 and
calcineurin (Cnb1) is hypersensitive to high levels of Ca2+
(17). CAX1-expressing K667 cells grow well in standard medium, but do
not grow in medium containing high levels of Ca2+ (12).
Only expression of deregulated CAX1 (containing mutations or
truncations of the N terminus) can suppress the yeast vacuolar Ca2+ transport defect (4, 13). Using a yeast expression
Arabidopsis cDNA library (19), we have isolated
Arabidopsis cDNAs that allow full-length CAX1-expressing
K667 strains to grow in medium containing high levels of
Ca2+. The cDNAs were isolated from the yeast and then
retransformed into K667 strains with or without CAX1 to verify that
they do not suppress the Ca2+ sensitivity of K667 by
themselves and to confirm that the clones are indeed required for CAX1
to suppress the Ca2+ sensitivity of K667. We have used the
term CXIP (CAX-interacting protein) for the protein products of these cDNAs.
Six different CXIP cDNA clones have been identified
using this yeast activation approach (Table
I). One of the cDNA clones, CXIP1, which is the focus of this study, encodes a novel
Arabidopsis protein with sequence homology at the C terminus
to PICOT proteins (24-26). We cloned an Arabidopsis homolog
of CXIP1 that we have termed CXIP2, which encodes
a protein of 293 amino acids (32 kDa). The yeast activation approach
also identified CXIP3, which encodes an FKBP protein
(A. thaliana FKBP15-2) (27). Four of the six cDNA
clones identified in the yeast activation screen are identical and
encode a protein whose function has not been previously characterized. We have termed this gene CXIP4.
K667 yeast cells expressing CXIP1 or CAX1 alone were unable to grow on
200 mM CaCl2, whereas coexpression of CXIP1
with CAX1 significantly enhanced the growth of K667 (Fig.
1A). However, growth of the
(CAX1 + CXIP1)-expressing strain was not as strong as growth of K667
cells expressing N-terminally truncated sCAX1. The ability to suppress
the Ca2+ sensitivity of the yeast mutants inferred
activation of autoinhibited CAX1; however, it was important to
demonstrate directly through biochemical studies that the combination
of full-length CAX1 and CXIP1 mediated H+/Ca2+
antiport activity in yeast. Therefore, we measured
The CXIP1 cDNA consists of 522 base pairs
(GenBankTM/EBI accession number AY157988). The amino acid
sequence consists of 173 amino acids (with a predicted molecular mass
of 19.3 kDa) and has extensive homology to PICOT proteins found in a
broad taxonomic distribution, including mammals, yeast, and bacteria (28). In other organisms, these proteins appear to play a negative regulatory role in cellular stress responses (24, 29). The primary
amino acid sequence of CXIP1 (Fig. 2) is
26% identical (38% similar) to the 171-amino acid GLP-1 PICOT protein
from Plasmodium falciparum (PfGLP-1) and 33%
identical (45% similar) to the 335-amino acid human PICOT
(HsPICOT) protein. The PICOT proteins contain a modular unit
of 84 amino acid located in the C terminus that is highly conserved
(PICOT-HD) (Fig. 2). The human PICOT protein has two tandem PICOT-HD
repeats, whereas both CXIP1, CXIP2, and P. falciparum GLP-1
have a single PICOT-HD in their C termini. Within the PICOT-HD, several
stretches of amino acids appear to be highly conserved, for example,
-CGFS- and -SNWPT- (amino acids 97-100 and 133-137, respectively)
(Fig. 2).
Among the various PICOT proteins, several include a single thioredoxin
(Trx) HD in their N termini. The human PICOT protein is involved in the
interaction with protein kinase C through this Trx-HD and negatively
regulates the c-Jun N-terminal kinase/AP-1 and NF- Specificity of CXIP1--
Using the yeast Ca2+
suppression assay, we were interested in determining whether CXIP1 and
CXIP2 can activate various CAX transporters. Three close homologs of
CAX1 (CAX2-4) also appear to be N-terminally regulated, and neither
full-length CAX3 nor CAX4 is able to suppress the Ca2+
sensitivity of K667 (10, 30). Unlike CXIP1, CXIP2 could not activate
CAX1 (Fig. 3A). CXIP1 and
CXIP2 could both weakly activate full-length CAX4 (Fig. 3B);
however, both CXIP1 and CXIP2 failed to activate CAX2 1
and CAX3 (data not shown).
Properties of CXIP1--
As an initial step toward deducing the
mechanism of CAX1 activation by CXIP1, we tested the ability of CXIP1
to activate various chimeric CAX constructs. CAX3 could not suppress
the K667 Ca2+-sensitive phenotype. In fact, an N-terminal
truncation of CAX3 (sCAX3) (21) was also unable to suppress the
Ca2+ toxicity despite lacking its autoinhibitory domain
(Fig. 4A). Just as CXIP1 was
unable to activate full-length CAX3, CXIP1 coexpressed with sCAX3 was
unable to suppress the yeast Ca2+ sensitivity (Fig.
4A). Numerous sCAX3-CAX1 constructs + CXIP1 were unable to
suppress the Ca2+ sensitivity of K667 yeast cells. For
example, K667 strains in which CXIP1 was coexpressed with chimeric
sCAX3-CAX1 constructs containing the central region or C terminus of
CAX1 were unable to suppress the Ca2+-sensitive phenotype
(Fig. 4, B and C). However, when a chimeric construct (called sCAX3- CXIP1 Associates with the N Terminus of CAX1 in Yeast--
To
determine whether this activation of CAX1 by CXIP1 is caused by a
physical interaction between CXIP1 and the CAX1 N terminus, a yeast
two-hybrid experiment was performed. CXIP1 coexpressed with the N
terminus of CAX1 (Met37-Leu73 or
Met1-Asn65) caused the lacZ gene in
the Y190 yeast strain to be activated, indicating that a direct
interaction between CXIP1 and the CAX1 N terminus had occurred (Fig.
5A). No color reaction
occurred in the absence of CXIP1 or in the absence of the CAX1
N-terminal fragments.
We have previously demonstrated that a synthetic peptide corresponding
to the first 36 amino acids of the CAX1 N terminus is able to inhibit
H+/Ca2+ transport mediated by N-terminally
truncated CAX1 (sCAX1), but does not inhibit Ca2+ transport
by other truncated H+/Ca2+ antiporters (13).
The interaction of this N-terminal peptide with CAX1 was mapped to
include residues 56-62 within CAX1 (13). We were interested in
coexpressing CXIP1 and sCAX1 to determine whether the presence of CXIP1
would alter the amount of synthetic peptide needed to inhibit
N-terminally truncated CAX1. A peptide concentration of 5 ± 1.5 µM was sufficient to inhibit 50% of 10 µM
Ca2+ transport activity mediated by sCAX1 (Fig.
5B) (13), whereas 12 ± 2 µM peptide was
required to inhibit 50% of sCAX1 transport in the presence of CXIP1
(Fig. 5B).
Disruption of the CXIP1 PICOT-HD--
To determine whether the
CXIP1 PICOT-HD is required for activation of full-length CAX1, we
mutated two highly conserved regions within the PICOT-HD (Fig. 2). The
conserved CGFS domain (amino acids 97-100) was changed to AAAA, and
the SNWPT domain (amino acids 133-137) was changed to AAAAA. Growth of
CAX1-expressing K667 cells on 200 mM CaCl2 was
virtually absent in the presence of either of the CXIP1 mutants (data
not shown).
Expression of CXIP1--
Northern blot analysis was used to assess
the expression of CXIP1 mRNA in various
Arabidopsis tissues. Hybridization with the CXIP1
probe revealed expression of CXIP1 in all tissues, with abundant expression in leaves and low level expression in roots and
flowers, compared with almost equal expression of CAX1 in leaf, stem, and flower tissue and very low expression in roots (Fig.
6). When seedlings were exposed to
different metals, there were alterations in the expression of
CXIP1. CXIP1 levels were modestly increased when
Ca2+ was added to the medium and decreased when
Na+, Mn2+, and Ni2+ were
added to the medium (Fig. 6).
Using a yeast-based approach to activate full-length CAX1, we have
identified an Arabidopsis cDNA termed
CXIP1 gene, encoding a predicted protein containing a
PICOT domain (Figs. 1 and 2). This domain is highly conserved
throughout evolution, and comparative sequence analysis indicates that
the PICOT-HD is distinct from all protein domains (28). The
physiological function of the PICOT-HD has not been identified; and
until this study, no plant gene with this motif has been functionally
characterized. In this work, we have demonstrated that, in a yeast
expression system, CXIP1 interacted with the N terminus of CAX1 to
modify vacuolar H+/Ca2+ antiport activity. This
study thus ascribes, for the first time, a function to a plant gene
containing a PICOT-HD. Like all PICOT-HD-containing proteins, CXIP1
appears to be a soluble protein. Two other non-transmembranous proteins
that activated CAX1 Ca2+ transport in yeast were also
identified in this study (Table I). CXIP3 is identical to a previously
characterized protein, A. thaliana FKBP15-2, a member of the
FKBP-type immunophilin family (27), whereas CXIP4 is a novel protein of
unknown function that appears to be unique to plants. We chose to
confine this study to further analysis of CXIP1.
Several proteins that contain a PICOT-HD also contain a Trx-HD,
including Arabidopsis CXIP2, which weakly activated
CAX4 Ca2+ transport (Fig. 3B). Proteins
containing the Trx-HD are important in a range of cellular process,
including controlling the redox state of the cell (31). In plant
Ca2+ signaling, there have been some reports linking the
plasma membrane redox state with release of Ca2+ from
intracellular stores (32); however, the mechanisms for transducing
these signals have not been elucidated.
The inability of CXIP2 to activate CAX1 suggests specificity in the
CXIP1-CAX1 interaction. Three different experimental approaches suggest
this specificity depends on the N terminus of CAX1. Two-hybrid analysis
demonstrated an interaction between CXIP1 and the N terminus of CAX1
(Fig. 5A). This interaction did not require the first 36 amino acids of CAX1, as a construct containing
Met37-Leu73 interacted with CXIP1 in a manner
indistinguishable from the CAX1 Met1-Asn65
construct. The second approach utilized chimeric CAX3 constructs to
demonstrate the importance of the N terminus of CAX1. Only the chimeric
CAX3 construct containing Met37-Leu73 from
CAX1, but lacking the first 36 amino acids of CAX1 (sCAX3- The requirement for the CXIP1 PICOT-HD in this interaction was inferred
by the inability of mutant forms of CXIP1 to activate CAX1 (data not
shown). However, at this time, we cannot rule out the possibility that
these mutants perturb protein expression or stability. Computer
analysis suggests that the PICOT-HD may form a globular topology, which
includes three We suggest that CXIP1 may activate CAX1 through a direct interaction
that disrupts autoinhibition and thus alters the confirmation of
autoinhibited CAX1 to allow Ca2+ transport. However, the
Ca2+ transport mediated by CXIP1-activated CAX1 is much
less than that of deregulated sCAX1 (Fig. 1). Evidence suggests that
activation of CAX1 by N-terminal truncation (as with sCAX1) is
artificial and does not occur in planta (11), and we believe
that the strong Ca2+ transport activity mediated by sCAX1
may not be realized by fully activated CAX1. For example,
Ca2+ transport mediated by a version of full-length CAX1
activated by point mutations within the N terminus is less than that
meditated by sCAX1.1 Similarly, Ca2+ transport
mediated by the calmodulin-activated Arabidopsis
Ca2+-ATPase ACA2 is significantly less than that mediated
by an N-terminally truncated version of ACA2 lacking its
calmodulin-binding autoinhibitory domain (33). However, the level of
H+/Ca2+ antiport activity measured for
CXIP1-activated CAX1 is still much less than the activity measured in
Arabidopsis tissue. It is possible that additional protein
interactions, such as with a multiple protein complex, are required to
fully activate CAX1. Other proteins may be required to interact
directly with CAX1 or cooperatively with CXIP1, or CXIP1 itself may
require additional regulation.
Interestingly, it appears that CXIP1 is able to strongly activate
some chimeric CAX constructs. The level of Ca2+ transport
measured in yeast vesicles expressing CXIP1-activated sCAX3- Previously, we demonstrated that truncations or additions to the N
terminus of CAX1 or CAX4 can activate these Ca2+
transporters in yeast (10). Similarly, CAX3 can also be activated by
the addition of amino acids to the N terminus (10). These findings
imply that any protein binding to the N-terminal region could activate
CAX1 transport in yeast by altering the autoinhibitory region or the
conformation of the transporter. Thus, although CXIP1 clearly interacts
with CAX1 at the N terminus in our various yeast assays, we cannot
discriminate between gene products that activate or repress CAX1
transport in plants.
The expression pattern of CXIP1 RNA mirrors the
expression pattern of CAX1 in plants (Fig. 6). That is, they
were both expressed in all tissues; both were modestly induced by
exogenous Ca2+; and both were repressed by certain ions.
Interestingly, aside from the low level expression of CXIP1
in flowers, the gene appears to be expressed at higher levels than
CAX1. The expression patterns of CXIP3 (A.
thaliana FKBP15-2) and CXIP4 were both also induced by
Ca2+ and regulated by different ions (data not shown);
however, the expression levels and tissue distribution of these genes
differed from those of CXIP1, suggesting that those
CXIP genes might play different roles in the regulation of
CAX1 activity in planta.
The use of yeast to reconstitute a plant response pathway has been
achieved with genes involved in Na+ homeostasis (34).
However, these genes were first identified using standard genetic
approaches (35). Here we have demonstrated that CXIP1, but not its
homolog CXIP2, can regulate the Ca2+ transport activity of
CAX1 in yeast. Furthermore, CXIP1 and CXIP2 may be involved in the
regulation of another putative H+/Ca2+
antiporter, CAX4. Indeed, we have previously proposed that each CAX
transporter may be regulated by different pathways (30). We speculate
that multiple components (or CXIPs) are required for regulation of CAX1
and other plant CAX transporters and that each CXIP may differ in its
interaction with CAX1. Future work using reverse genetics in
Arabidopsis will help clarify the contribution of CXIPs to
CAX-mediated H+/Ca2+ transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ade2-1
can1-100 his3-11,15 leu2-3,112
trp1-1 ura3-1) (17) was used in all yeast experiments
involving the expression of CAX genes with or without
CXIP (CAX-interacting
protein) genes. Yeast strain Y190 (MATa
gal4 gal180 his3 trp1-901 ade2-101 ura3-52
leu2-3,112 + URA3::GAL
lacZ, LYS2::GAL(UAS)
HIS3 cyhr) was used in the yeast two-hybrid assay
(18). The A. thaliana Columbia ecotype was used in this study.
2 × 105 colonies were replicated onto yeast
extract/peptone/dextrose (YPD) medium supplemented with 200 mM CaCl2 to identify Ca2+-tolerant
transformants. cDNA clones of interest were isolated, and plasmid
DNA was retransformed into the CAX1-expressing K667 yeast cells to
confirm that the activation of CAX1 Ca2+ transport activity
was attributable to the Arabidopsis cDNA inserts. All
cDNA inserts were subcloned into pBluescript and completely sequenced.
, sCAX3-
, sCAX3-
, sCAX1-
, sCAX3-
1, and
sCAX1-9 were constructed in a previous study (21). Amino acid
substitutions were introduced into the CXIP1 PICOT (protein
kinase C-interacting cousin of
thioredoxin) homology domain
(HD)2 using a modified
phoenix mutagenesis protocol with the Type IIS restriction enzyme
BsmBI (22). To change amino acids CGFS to AAAA, forward
primer 5'-GAA TCC CGT CTC CCC ATG GCT GCG GCA GCC AAC ACT
GTG GTT CAG ATT TTG-3' (where italic letters indicate the mutated
residues) and reverse primer 5'-G AAA GGA ACG AGA GAC TTC CCC ATG GAG
ACG GAA TTC-3' were used. To change amino acids SWNPT to AAAAA, forward
primer 5'-GAA TCC CGT CTC GAG TAT GCT GCA GCA GCC GCG TTT
CCT CAG CTT TAT ATC-3' was used along with reverse primer 5'-TTG AGG
CAA GGA CTT AAA GAG TAT GAG ACG GAA TTC-3'. All mutant constructs were
completely sequenced and subcloned into the yeast expression vector
piHGpd (23). mRNA transcript expression in yeast cells was
confirmed by reverse transcription-PCR (24).
80 °C for an appropriate time.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of CXIPs
pH-dependent 10 µM
45CaCl2 uptake into microsomal vesicles
isolated from K667 yeast strains expressing both Arabidopsis
cDNAs. H+/Ca2+ transport activity was
observed in membrane vesicles from a (CAX1 + CXIP1)-expressing strain
and an sCAX1-expressing strain (Fig. 1B). However, no
H+/Ca2+ antiport activity was detectable in
vesicles from full-length CAX1-expressing or CXIP1-expressing yeast
strains (Fig. 1B and data not shown). The
H+/Ca2+ antiport activity measured in CAX1 + CXIP1 vesicles was consistently ~10% of the activity measured in
sCAX1 vesicles.
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Fig. 1.
CXIP1 activates CAX1 Ca2+
transport activity. A, suppression of the
pmc1vcx1cnb1 yeast Ca2+ sensitivity in sCAX1-
and (CAX1 + CXIP1)-expressing cells. Yeast cells coexpressing various
plasmids, as indicated, were grown to A650 = 1.0 in selection medium at 30 °C. Cells were diluted 5-fold, and 5 µl
of each dilution was spotted onto selection medium (lacking His and
Ura) and YPD medium supplemented with 200 mM
CaCl2. Photographs were taken after 3 days. B,
uptake after 12 min of 10 µM
45Ca2+ into endomembrane vesicles prepared from
S. cerevisiae strain K667 after transformation with sCAX1,
CAX1 + CXIP1, and CAX1. Uptake was estimated as the difference between
parallel samples with and without the addition of gramicidin (5 µM). Data are the means of three independent experiments,
and the bars indicate S.E.
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Fig. 2.
CXIP1 is an evolutionarily conserved PICOT
domain-containing protein. Shown is an alignment of deduced amino
acid sequences of CXIP1 with other PICOT domain-containing proteins
encoded by the Arabidopsis CXIP1 homologous gene
(CXIP2), the human PICOT (HsPICOT) gene, and the
GLP-1 gene from P. falciparum
(PfGLP-1). Alignments were performed using the ClustalW
Version 1.8 program (Baylor College of Medicine) (36). Consensus amino
acid residues are boxed in black (identical) or
gray (similar). Gaps introduced to maximize the alignments
are denoted by dashes. The PICOT domains located in the C
terminus are overlined. The asterisks indicate
the conserved amino acid sequences within the PICOT domain. The protein
and DNA accession numbers for CXIP1, CXIP2, human PICOT, and P. falciparum GLP-1 are AY157988, AY157989, AAF28844, and AAK00581,
respectively.
B pathway (24).
CXIP2 is 30% identical (43% similar) to CXIP1 overall and is >54%
identical (71% similar) to CXIP1 within the PICOT-HD. CXIP2 contains a
Trx-HD in the N terminus like that of the human PICOT protein (Fig.
2).
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Fig. 3.
CXIP1 and CXIP2 differentially activate CAX
transporters. A and B, K667 yeast cells
coexpressing various plasmids, as indicated, were grown as described in
the legend to Fig. 1.
1) was used that contained 37 amino
acids of the N-terminal region of CAX1
(Met37-Leu73) fused to CAX3, CXIP1 could
activate this chimeric construct (Fig. 4C). We have
previously shown that a 9-amino acid domain within CAX1, which we
termed the "Ca2+ domain," is required for
Ca2+ transport (21). Furthermore, when this 9-amino acid
domain from CAX1 is swapped into an N-terminally truncated CAX3
construct (to give sCAX3-9), this "activated" CAX3 mutant can
transport Ca2+ (21). The activation of CAX3 (sCAX3-
1) in
the presence of CXIP1 demonstrated in Fig. 4C did not
require the CAX1 9-amino acid Ca2+ domain. The chimera
sCAX3-
1, when coexpressed with CXIP1 in K667 yeast strains,
demonstrated
pH-dependent 10 µM
45CaCl2 uptake into microsomal vesicles (Fig.
4D). This H+/Ca2+ antiport activity
was comparable to that measured for sCAX1 (data not shown). The
sCAX1-CAX3 chimera sCAX1-9 (21), with the 9-amino acid Ca2+
domain removed and replaced with the equivalent 9-amino acid region of
CAX3, was not able to strongly suppress the Ca2+
sensitivity of K667; however, expression of CXIP1 + sCAX1-9 strongly suppressed the Ca2+ toxicity (Fig. 4C).
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Fig. 4.
Identification of domains in CAX1 that can
activate CAX3. A-C, suppression of the yeast vacuolar
Ca2+ transport defect mutant by CAX chimeras activated by
CXIP1. K667 yeast cells coexpressing various plasmids, as indicated,
were grown as described in the legend to Fig. 1. D,
Ca2+ uptake assay. Shown is a time course of 10 µM 45Ca2+ transport into
endomembrane-enriched vesicles prepared from yeast strains coexpressing
sCAX3- 1 and CXIP1. 45Ca2+ transport was
measured in the presence (
) and absence (
) of 5 µM
gramicidin. The Ca2+ ionophore A23187 was added at 12 min
to a concentration of 5 µM (indicated by the
arrow). All results shown here are the means of two to three
independent experiments, and the bars indicate S.E.
View larger version (23K):
[in a new window]
Fig. 5.
Interaction of CXIP1 with the N terminus of
CAX1. A, interaction of CXIP1 with the CAX1 N terminus
by yeast two-hybrid analysis. Y190 yeast cells coexpressing CXIP1 in
pACT2 and the CAX1 N-terminal fragments (CAX1-N(37-73aa) and
CAX1-N(1-65aa)) in pAS2 were grown on synthetic complete medium
lacking His, Trp, and Leu and assayed for LacZ expression. Shown are
the results from -galactosidase assays on a filter. B,
effect of the CAX1-N-terminal regulatory region peptide on
pH-dependent 10 µM
45Ca2+ transport by sCAX1 (
) and sCAX1 + CXIP1 (
) into yeast endomembrane vesicles measured at a 10-min time
point with various concentrations of CAX1-N-terminal regulatory
region peptide. The results are shown following subtraction of
the gramicidin background values and as a percentage of
Ca2+ uptake of the control sample in the absence of
peptide. All results shown here are the means of three independent
experiments, and the bars indicate S.E.
View larger version (36K):
[in a new window]
Fig. 6.
Tissue distribution and induction of CAX1 and
CXIP1. A, total RNA samples were isolated from
Arabidopsis rosette leaves (L), stems
(St), roots (R), and flowers (Fl).
B and C, total RNA samples were extracted from
3-week-old Arabidopsis seedlings previously treated for
12 h with the following solutions: water (as a control), Murashige
and Skoog (MS) nutrient medium, 10 mM
CaCl2, 80 mM NaCl, 2 mM
MnCl2, and 0.1 mM NiSO4. Twenty
micrograms of total RNA was hybridized with gene-specific probes
for CAX1 and CXIP1. EtBr-stained rRNAs are shown as a
loading control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1), was
capable of being activated by CXIP1 (Fig. 4, C and
D). The third approach utilized a competition experiment to
suggest that CXIP1 binds to the N terminus of CAX1. Our previous
studies have demonstrated that a synthetic peptide corresponding to
amino acids 1-36 of CAX1 specifically inhibits Ca2+
transport mediated by sCAX1 lacking these 36 N-terminal amino acids
(13). Furthermore, we showed that these 36 amino acids physically interact with amino acids 56-62 at the N terminus of CAX1
to facilitate autoinhibition of CAX1 (13). We hypothesized that if
CXIP1 binds to this same region (amino acids 56-62) of CAX1, more
peptide should be required to cause Ca2+ transport
inhibition. Indeed, approximately double the peptide concentration was
needed to inhibit 50% of sCAX1 transport activity in the presence of
CXIP1 (Fig. 5B).
-helices and intervening
-strands (26). It has
been proposed that this structure can (i) form a transient
intermolecular interaction; (ii) bind to substrates, regulators, or
cofactors; or (iii) tether proteins to specific subcellular
compartments. In the future, it will be interesting to further
delineate the mechanism of CXIP1-mediated CAX1 regulation.
1 was
equivalent to the level of activity in vesicles expressing sCAX1 (Fig.
4D). Our previous findings show that the sCAX1-CAX3 chimera sCAX1-9 is unable to transport Ca2+ (21).
This construct contains an N-terminal truncation, but also contains the
9-amino acid region from CAX3 (residues 87-95) that replaced the
equivalent 9-amino acid Ca2+ domain of CAX1 and was thought
to abolish function due to the inability of this CAX3 domain to allow
Ca2+ transport. However, we show here that sCAX1-9 could
also be activated by coexpression of CXIP1 and that this activity was
equivalent to that of sCAX1 (Fig. 4C).
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ACKNOWLEDGEMENTS |
---|
We thank Sherry LeClere and Jon Pittman for comments.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Department of Agriculture/Agricultural Research Service Cooperative Agreement 58-6250-6001 and by National Institutes of Health Grants CHRC 5 P30 and 1R01 GM57427.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY157988, AY157989, and AY163162.
To whom correspondence should be addressed: USDA/ARS
Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030. Tel.: 713-798-7011; Fax: 713-798-7078; E-mail: kendalh@bcm.tmc.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210883200
1 J. K. Pittman and K. D. Hirschi, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HD, homology domain; Trx, thioredoxin.
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