From the Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zürich, Switzerland and the Department of Biological Chemistry, University of Padova, 35121 Padova, Ialy
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ABSTRACT |
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Calcium-pumping ATPases are an essential
component of the intracellular calcium homeostasis system and have been
characterized in a large variety of species and cell types. In
mammalian genomes, these proteins are encoded by gene families whose
individual members feature complex tissue-specific expression and
alternative splicing. In the search for a less complex system that is
more amenable to genetic manipulation, we have identified a family of
three genes (mca-1, mca-2, and
mca-3) encoding putative calcium ATPases in the
Caenorhabditis elegans Genome Project data and completed their transcript structure. In this work, we report the cloning and
functional expression of the mca-1 gene, which encodes a
calcium-stimulated ATPase whose features resemble those of the plasma
membrane calcium adenosine triphosphatase family of mammalian cells and
appears to be regulated by a multipartite promoter.
Calcium-pumping ATPases have been characterized from a vast
variety of eukaryotic organisms. Sequence analysis of more than 40 complete cDNA sequences and biochemical investigations of them have
defined two major classes of these proteins: (a) those
residing on the plasma membrane
(PMCA),1 which are stimulated
by interaction with calmodulin (for reviews, see Refs. 1-3), and
(b) organellar ATPases (SERCA), located at various
intracellular membranes (for a review, see Refs. 4 and 5). The
complexity of these protein families makes their study difficult,
stressing the advantages of more accessible unicellular models that are
amenable to genetic manipulation, such as yeast (for a review, see Ref.
6). Whereas the yeast model has contributed to the advancement of our
understanding of the roles calcium pumps play in various cellular
processes (7-10), the yeast cell, with its rigid wall, is closer to
plants in that a major calcium storage organelle appears to be vacuole,
rather than sarcoplasmic reticulum. This limits the projection of the
yeast model to understanding the role of these proteins in a mammalian
cell. Increasing evidence that the plasma membrane-based calcium
transporters reside at specific subcellular structures, such as the
caveolae (reviewed in Ref. 11), and other differences between animal
and plant cell architecture demand a more adequate model of an animal
cell than the yeast cell. One may add that despite substantial
conservation of the protein structure, the modes of gene regulation of
mammalian and yeast ATPase genes are likely to have very little in
common. The study of the transcriptional regulation of PMCA genes is a technically challenging task (12) that is unlikely to be approached systematically before the sequence of at least one mammalian genome becomes available. Moreover, in mammalian systems, promoter studies are
limited to tissues that are amenable to temporal cultivation and
transient transfection. Whereas solitary examples of PMCA gene
overexpression (13) and knockout (14) do exist, comprehensive genetic
manipulation of mammalian families remains extremely time and labor intensive.
The nematode Caenorhabditis elegans is an established model
for developmental and neurogenetic studies and is the first
multicellular organism whose genome sequence is soon to be completed.
More than half of the C. elegans genes have a significant
match to a mammalian gene with a similar or identical function
(reviewed in Ref. 15). Thus, it represents a unique model for studies
of calcium homeostasis in a genetically manipulatable multicellular
organism. Voltage-gated calcium channels (16, 17), ryanodine receptors
(18, 19), and inositol 1,4,5-triphosphate receptors (20) have already been found in C. elegans. A putative SERCA-type pump was
cloned from the parasitic nematode Schistosoma mansoni (21).
In contrast to mammalian systems, an essentially complete catalog of
candidate calcium transporters will be derived from genomic sequencing
data in the near future. As a step toward this goal, we have
characterized a family of C. elegans calcium ATPases, which
appear to be similar to the mammalian PMCA family and are apparently
regulated by multipartite promoters.
Isolation of cDNA Clones, Sequencing, and Computer
Analysis--
BLAST (22) searches of the Wormpep database with the
amino acid sequence CSDKTGTLT were run on the C. elegans
World Wide Web server at the Sanger Center (Cambridge, United Kingdom).
Clones identified in the EST database were clustered using the
Sequencher program (Gene Codes, Ann Arbor, MI) and further analyzed
with this program and with the GCG package v.8.0 (23). Clones with the
largest cDNA inserts for each cluster were obtained from Y. Kohara
(Institute of Genetics, Mishima, Japan) and sequenced using IR800-labeled dye primers on a Li-Cor 4000L automated sequencer. Sequence analyses were run through the World Wide Web interface at the
Swiss Bioinformatics Institute (http://www.isb-sib.ch/).
Isolation of Total RNA, cDNA Synthesis, and Reverse
Transcription-PCR--
Strain N2 was obtained from the
Caenorhabditis Genetics Stock Center and propagated
according to established methods. Total RNA was isolated from about 500 mg of worms using Trizol reagent (Life Technologies AG, Basel,
Switzerland). 250 µg of total RNA were used to prepare polyadenylated
RNA using Oligotex columns according to the manufacturer's procedures
(Qiagen AG, Basel, Switzerland). RNA was eluted in 20 µl of water,
split into two portions, and immediately used to synthesize cDNA
using Expand reverse transcriptase (Boehringer Mannheim AG, Rotkreuz,
Switzerland) and either oligo dT20-Not primer or hexamer primer
(Pharmacia Biotech AG, Dubendorf, Switzerland). The completed cDNA
reaction was kept at 4 °C and was typically used up within a month.
Reverse transcription-PCR was carried out using the Expand Long
Template PCR kit (Boehringer Mannheim) with one of several
gene-specific primers and SL1-specific primer
5'-CGCGGGTTTAATTACCCAAGTTTGAG.
Construction of the Full-Length Transcript and Its Expression in
Insect Cells--
A PCR product extending from the penultimate base
preceding the AUG codon to the unique AvaI site was prepared
from cDNA and joined to the AvaI-cut plasmid clone
yk49f8 (obtained from Y. Kohara). To introduce a FLAG-tagging sequence,
the fragment between the unique PshAI site in the cDNA
and the XbaI site after the stop codon was replaced by a
double-stranded synthetic oligonucleotide, coding for the respective
C-terminal sequence (VDMEDIELN) followed by the FLAG sequence, a new
stop codon, and a XbaI site. The correct clone was selected
by DNA sequencing. A pFastBac clone with the FLAG-tagged cDNA was
transferred into the bacmid, following the instructions of the
baculovirus expression kit manufacturer (Bac-to-Bac; Life
Technologies). The positive clone DNA was quantitated by fluorometry
and introduced into Sf9 cells essentially following the
instructions supplied with the transfection reagent (Lipofectin; Life
Technologies). The cells were allowed to recover in TNM FH medium
containing 10% fetal calf serum and 50 µg/ml hygromycin (Sigma) for
72 h, and the virus-containing medium was collected. After a
second infection with the virus, the cells were grown in the same
medium and harvested after 48 h. Crude membranes were prepared
essentially as described previously (24). Aliquots of the membranes (2 µl) were analyzed on 5% Protogel gels (National Diagnostics,
Atlanta, GA) containing the buffer system of Laemmli (25) and
electroblotted (26) onto a reinforced nitrocelulose membrane (OptiTran
BA-S 85; Schleicher and Schull, Dassel, Germany). The blots were probed
with the monoclonal antibody Anti-FLAG M2 (Kodak Scientific Imaging
Systems, Rochester, NY) at a 1:5000 dilution using the ECL Western kit
(Amersham Switzerland).
Phosphointermediate Formation of the ATPase--
Aliquots of the
crude membrane preparation (30 µg of protein) were incubated with
assay solutions containing either various cations (100 µM
CaCl2, 100 µM LaCl3, or both) or
5 mM EGTA and 0.3 µM
[ Calmodulin Binding Assay--
Membrane proteins of Sf9
cells were resolved by electrophoresis in 5% Protogel gels and
electroblotted onto Immobilon-P membrane (Millipore, Bedford, MA).
After electroblotting, the membrane was cut into strips containing
three tracks each (membrane proteins from untransfected cells, cells
transfected with the mca1 construct, and cells transfected
with the human pmca4 construct (31), respectively) and
washed twice with Ca-Tris-buffered saline (25 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, and 200 µM CaCl2). The strips were incubated for
1 h in the blocking solution (Ca-Tris-buffered saline supplemented with 3% gelatine and 0.05% Tween 20), washed twice with TBST buffer (Ca-Tris-buffered saline, supplemented with 0.05% gelatine and 0.05%
Tween 20), and incubated with biotinylated calmodulin dilutions in TBST
buffer at a concentration ranging from 10 nM to 1 µM for 2 h at room temperature. One of the strips
was incubated with 1 µM calmodulin as described above,
but 200 µM CaCl2 in all buffers was
replaced with 6 mM EGTA. Strips were subsequently
washed three times with TBST buffer, and biotin binding was visualized
using a streptavidin-alkaline phosphatase conjugate (Promega, Madison, WI) at a 1:1000 dilution, essentially as suggested by the manufacturer of the conjugate.
Identification of a Calcium ATPase Gene Family in C. elegans--
Systematic searches of C. elegans databases
available on-line from the Sanger Center have identified three sets
(Table I) of relatively abundant EST
clones with a high degree of similarity to mammalian calcium ATPases of
the PMCA family (Fig. 1). These sequences
are located on C. elegans chromosome IV and show about 70%
similarity to each other. Clones were obtained from the Institute of
Genetics (Mishima, Japan) and completed by reverse transcription-PCR, taking into account the fact that two-thirds of the C. elegans mRNAs are trans-spliced to the same
22-nucleotide leader SL1 (28, 29). In this experiment, however, it was
found that the transcripts always contained alternative 5' ends, only
one of which was predicted by GeneFinder scanning of the genomic
sequence. In one case, examination of the available genomic sequence
(cosmid W09C2) upstream of the predicted exon 2 (Fig.
2A) resulted in the
unambiguous localization of all four alternative 5' ends, which are
followed by a consensus splicing signal. Thus, the transcription of
this gene is apparently driven by a multipartite promoter, which is
spread over 4 kilobases. This analysis also showed that every
alternative exon appended an amino acid sequence to the basic form of
the protein, which is encoded in exon 2 and in the exons downstream.
The shortest form of mca-1 cDNA was used in the
expression experiments.
Expression of the Complete Transcript W09C2.3 in Insect Cells
Confirms Its Identity to a P-type Calcium ATPase--
The shortest
form, which was encoded in exons 2-10 (Fig. 2A), was
modified by PCR to include a FLAG-tagging sequence at the C terminus,
inserted in baculovirus vector pFast Bac, and introduced into the
Sf9 insect cells by lipofection. A crude membrane preparation from infected cells showed a new protein band of ~130 kDa that bound
monoclonal antibody M2 (Fig.
3A), confirming that the
full-length open reading frame W09C2.3 was expressed in Sf9
cells. A crude membrane preparation from baculovirus-infected cells was
then subjected to the functional assay (30) for P-type ATPases; in the
case of the PMCA pump, the rapid degradation of the intermediate can
typically be slowed down by lanthanum, allowing its detection in acidic
gel systems (27). The results of the assay showed that a clear signal
in the 130-kDa area could be detected in infected cells, even if the
expression level of the new protein was low. The signal was not present
in the control counterparts (Fig. 3B). The
phosphointermediate pattern, i.e. an extremely weak (if
any) reaction in the presence of calcium alone, the absence of
reaction in the presence of EGTA, and, especially, the increase in the steady-state level of the phosphointermediate in the presence of
lanthanum and calcium corresponded to that observed with pumps of the
PMCA family (24, 31). An endogenous P-type ATPase of 110-120 kDa was
also detected by the assay, but it was clearly Ca2+
independent (curiously, its phosphoenzyme formation was inhibited in
cells expressing the mca-1 construct). The nature of this
putative P-type ATPase, which is routinely observed in the assay on
Sf9 cells, is unknown.
Mca Gene Products Contain Calmodulin-binding Domains at their C
Termini--
Important features of the mammalian PMCA pumps, such as
the location and spacing between the transmembrane domains, the
identity of the sequence of the phosphorylation site (32) and of the FITC site (33) as well the presence of the C-terminal extension that
accounts for the molecular mass of 130 kDa, as compared with those of a
typical SERCA-type pump (34), all point to the conclusion that the Mca
family proteins belong to the PMCA family ATPases (Fig. 1). Whereas the
overall sequence identity between mammalian and nematode proteins at
the 200 C-terminal amino acids appears to be low, the most pronounced
region of similarity (Fig. 4A) corresponds to the location of the calmodulin-binding/autoinhibitory domain of the mammalian pumps (35, 36). In all three nematode sequences, this region of similarity features an obvious propensity to
form an amphiphilic helix, with clustering of hydrophobic residues on
one side, and a variable number of basic residues on the opposite side
(Fig. 4B). In a membrane-based assay, the expressed
mca-1 protein actually binds human calmodulin at a
concentration of 10 nM or higher (Fig.
5). This experiment clearly demonstrates the specific binding of human calmodulin to a protein, which is only
present in the cells, expressing the mca-1 construct.
Moreover, this protein is indeed expected to have a very similar
mobility to the expressed human PMCA4. Two kinds of control samples
used in this experiment sort out the interference of an endogenous streptavidin-binding protein (Fig. 5, esb), because this
band is the only one remaining in the presence of EGTA (not shown).
Finally, whereas a calmodulin-binding domain is not necessarily
identified by its sequence similarity to another one, in this case, a
significant consensus sequence can be derived (Fig. 4A), which suggests that the Mca family may actually contain orthologs of
the PMCA family, particularly because at a >90% completion of
the nematode genome, no other suitable candidates could be found.
The genome projects have resulted in the identification of
numerous proteins with a candidate function inferred from sequence similarity to known proteins and/or a genetically identified phenotype. In this work, database searches have revealed three groups of EST
clones showing about a 65% similarity to mammalian calcium ATPases of
the PMCA family. The location and spacing between the major sequence
motifs of the mca-1 protein unequivocally place it in the
PMCA family and rule out the possibility that it belongs to a
SERCA-type family or to any novel Ca2+-ATPase family not
found thus far in mammalian cells. In fact, a SERCA-type sequence is
encoded on chromosome III (cosmid K9D11), whereas all three
mca genes reside on chromosome IV. However, the
mca-1 gene has a number of features that are not found in pmca genes, namely, the presence of alternative splicing at
the extreme N terminus, the absence of internal splicing site A found in the pmca genes, and the absence of alternative splicing
around the calmodulin-binding
domain,2 as compared with the
mammalian alternative splicing site C (37, 38). A peculiar feature of
mca-1 protein (which is not found, however, in
mca-2 and mca-3) is the presence of the
serine-rich region, which includes 11 consecutive residues, located
between transmembrane segments 2 and 3. This region replaces the
stretch of alternating lysines and glutamic acid residues responsible for phospholipid sensitivity (39), which is pronounced in PMCA pumps
but absent in SERCA pumps.
The calmodulin-stimulated mammalian PMCA family has been the subject of
extensive studies during the last decade, after the cloning of the
first cDNAs in the late 1980s. However, whereas single cloned PMCA
isoforms could be expressed in a model system based on a cell line and
some of their biochemical properties could be studied (for a review,
see Ref. 3), the implication of their specific features at the
cellular/organismal level was beyond the possibilities of the
experimental system. The identification of nematode analogs of the
mammalian PMCA pumps now paves the way to reverse genetic experiments
on the organismal level.
At the time of writing, the C. elegans genome seems to
contain only three genes, showing substantial similarity to the
mammalian PMCA family as well as five candidate plasma membrane
Na/Ca(K) exchangers.2 Although it is not excluded that
other candidate genes still reside in the gaps of the genomic data, or
that they are missed in the EST data due to the toxicity of this
protein or portions thereof to the Escherichia coli cell,
the number and variety of candidate plasma membrane calcium
transporters in the nematode approach the level of complexity known for
mammalian systems. Experiments can now be designed to study the cell
specificity of expression using the transgenic reporter gene expression
approach (40, 41) and the results of systematic gene inactivation using the resources of a random knockout library produced in C. elegans with the use of the Tc1 transposable element (42, 43).
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]ATP (Amersham) in a volume of 50 µl,
essentially as described previously (13), and analyzed on 5% Protogel
containing the buffer system of Sarkadi et al. (27). The
gels also contained the Rainbow protein marker (Amersham). The gels
were dried on Whatman 3MM paper in a gel dryer at 65 °C for 1 h
and exposed to a storage phosphor screen (Kodak) for 2 h or
overnight. The screen was scanned in a STORM imager and analyzed with
ImageQuant 1.2 software (Molecular Dynamics, Sunnyvale, CA).
RESULTS
Candidate calcium ATPases found in the C. elegans Genome Project
data
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Fig. 1.
Alignment of the four human PMCA gene
products (hum_1 through hum_4) with the product of C. elegans open reading frame W09C2.3 (cemca_1). Black
shading shows the sequences that are identical in all five
proteins. Putative transmembrane domains are underlined and
numbered 1 through 10. Functional sites are underlined with
a thick line. The figure was compiled from the following
SwissProt/TrEMBL entries: P20020 (hum_1), P23634 (hum_4), Q01814
(hum_2), Q16720 (hum_3), and O45215 (cemca_1). Alignment with only one
nematode sequence is shown for clarity.
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Fig. 2.
Structure of the 5' terminus of the
mca-1 gene. A, reconstruction of the gene
structure and the splicing options at the 5' terminus by comparison of
the sequence data from the PCR products with the genomic sequence in
cosmid clone W09C2 (accession number Z68221). Gene-specific primers
(GSP) used for PCR are indicated by arrows.
B, nested reverse transcription-PCR amplification of the
alternative first exons of the mca-1 gene.
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Fig. 3.
Analysis of crude membrane proteins of
Sf9 cells expressing epitope-tagged mca-1
cDNA. A, Western blot analysis with the anti-FLAG
antibody. Lanes 1 and 2 contain the membranes
from the primary transfected cells (a weakly reactive band of 130 kDa
is seen at a longer exposure). Lanes 3a and 3b
contain membranes from two independent midscale recombinant virus
propagations. The lane with membranes from uninfected cells is labeled
Sf9. B, phosphointermediate assay for
P-type ATPases. S, membranes from uninfected cells;
O and N, two independent membrane preparations
from cells infected with the recombinant virus. The cation compositions
of the reaction media are indicated above the lanes.
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Fig. 4.
Putative calmodulin binding sites from the
Mca proteins. A, alignment with the sites of human PMCA
proteins. Shading indicates positions at which 50% of the
sequences match. B, a helical wheel representation of the
putative calmodulin-binding domains of the Mca proteins. Hydrophobic
amino acids are shown in shadow lettering.
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Fig. 5.
Binding of human calmodulin to the Mca1
protein expressed in Sf9 cells. Gel-fractionated and
electroblotted membrane proteins from untransfected cells (lane
U), cells transfected with the mca-1 construct
(lane CE), and cells transfected with the human
pmca4 construct (lane H4). Sf9 cells were
incubated with 10 nM biotinylated human calmodulin and 200 µM Ca2+. The band that is visible only in the
presence of Ca2+ is designated mca; the
endogenous streptavidin-binding protein is designated esb.
The numbers at the left show the positions of the
protein marker bands. The amount of protein loaded in lane
H4 was 3-fold lower than that in lanes U and
CE.
DISCUSSION
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Note Added in Proof |
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While this paper was in press, the genome sequence of C. elegans was completed (The C. elegans Sequencing Consortium (1998) Science 282, 2012-2018).
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FOOTNOTES |
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* 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/EMBL Data Bank with accession number(s) AJ223616, AJ010708, and AJ010646.
To whom all correspondence should be addressed: Laboratory of
Biochemistry III, Swiss Federal Institute of Technology (ETH) Universitätsstrasse 16, CH-8092 Zürich, Switzerland. Tel.:
41-1-632-30-11/12; Fax: 41-1-632-12-13; E-mail:
carafoli{at}bc.biol.ethz.ch.
The abbreviations used are: PMCA, plasma membrane calcium adenosine triphosphatase; SERCA, sarcoendoplasmic reticulum calcium adenosine triphosphatase; EST, expressed sequence tag; PCR, polymerase chain reaction.
2 Unpublished data.
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REFERENCES |
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