Identification and Functional Expression of the Plasma Membrane Calcium ATPase Gene Family from Caenorhabditis elegans

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, andmca-3) encoding putative calcium ATPases in theCaenorhabditis 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 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, andmca-3) encoding putative calcium ATPases in theCaenorhabditis 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), 1The abbreviations used are: PMCA, plasma membrane calcium adenosine triphosphatase; SERCA, sarcoendoplasmic reticulum calcium adenosine triphosphatase; EST, expressed sequence tag; PCR, polymerase chain reaction. which are stimulated by interaction with calmodulin (for reviews, see Refs. 1Carafoli E. J. Biol. Chem. 1992; 267: 2115-2118Abstract Full Text PDF PubMed Google Scholar, 2Carafoli E. FASEB J. 1994; 8: 993-1002Crossref PubMed Scopus (363) Google Scholar, 3Carafoli E. Garcia Martin E. Guerini D. Experientia (Basel). 1996; 52: 1091-1100Crossref PubMed Scopus (75) Google Scholar), and (b) organellar ATPases (SERCA), located at various intracellular membranes (for a review, see Refs. 4MacLennan D.H. Toyofuku T. Lytton J. Ann. N. Y. Acad. Sci. 1992; 671: 1-10Crossref PubMed Scopus (54) Google Scholar and 5MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). 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.6Catty P. Goffeau A. Biosci. Rep. 1996; 16: 75-85Crossref PubMed Scopus (21) Google Scholar). Whereas the yeast model has contributed to the advancement of our understanding of the roles calcium pumps play in various cellular processes (7Cunningham K.W. Fink G.R. J. Exp. Biol. 1994; 196: 157-166Crossref PubMed Google Scholar, 8Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar, 9Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (365) Google Scholar, 10Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (383) Google Scholar), 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. 11Monteith G.R. Roufogalis B.D. Cell Calcium. 1995; 18: 459-470Crossref PubMed Scopus (120) Google Scholar), 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 (12Hilfiker H. Strehler-Page M.-A. Stauffer T.P. Carafoli E. Strehler E.E. J. Biol. Chem. 1993; 268: 19717-19725Abstract Full Text PDF PubMed Google Scholar) 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 (13Guerini D. Schröder S. Foletti D. Carafoli E. J. Biol. Chem. 1995; 270: 14643-14650Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and knockout (14Kozel P.J. Friedman R.A. Erway L.C. Yamoah E.N. Liu L.H. Riddle T. Duffy J.J. Doetschman T. Miller M.L. Cardell E.L. Shull G.E. J. Biol. Chem. 1998; 273: 18693-18696Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) 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. 15Ahringer J. Curr. Opin. Genet. Dev. 1997; 7: 410-415Crossref PubMed Scopus (33) Google Scholar). Thus, it represents a unique model for studies of calcium homeostasis in a genetically manipulatable multicellular organism. Voltage-gated calcium channels (16Schafer W.R. Kenyon C.J. Nature. 1995; 375: 73-78Crossref PubMed Scopus (231) Google Scholar, 17Lee R.Y. Lobel L. Hengartner M. Horvitz H.R. Avery L. EMBO J. 1997; 16: 6066-6076Crossref PubMed Scopus (165) Google Scholar), ryanodine receptors (18Maryon E.B. Coronado R. Anderson P. J. Cell Biol. 1996; 134: 885-893Crossref PubMed Scopus (108) Google Scholar, 19Sakube Y. Ando H. Kagawa H. J. Mol. Biol. 1997; 267: 849-864Crossref PubMed Scopus (41) Google Scholar), and inositol 1,4,5-triphosphate receptors (20Baylis H. Furuichi T. Yoshikawa F. Mikoshiba K. Satelle D. European Worm Meeting. T-7. The Sanger Center, Cambridge UK1998Google Scholar) have already been found in C. elegans. A putative SERCA-type pump was cloned from the parasitic nematode Schistosoma mansoni (21de Mendonca R.L. Beck E. Rumjanek F.D. Goffeau A. Mol. Biochem. Parasitol. 1995; 72: 129-139Crossref PubMed Scopus (25) Google Scholar). 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. BLAST (22Altschul S.F. Gish W. Miller W. Myers E.W. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) searches of the Wormpep database with the amino acid sequence CSDKTGTLT were run on the C. elegansWorld 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 (23Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11531) Google Scholar). 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/). Strain N2 was obtained from theCaenorhabditis 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. 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 (24Heim R. Iwata T. Zvaritch E. Adamo H.P. Rutishauser B. Strehler E.E. Guerini D. Carafoli E. J. Biol. Chem. 1992; 267: 24476-24484Abstract Full Text PDF PubMed Google Scholar). Aliquots of the membranes (2 μl) were analyzed on 5% Protogel gels (National Diagnostics, Atlanta, GA) containing the buffer system of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and electroblotted (26Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar) 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). Aliquots of the crude membrane preparation (30 μg of protein) were incubated with assay solutions containing either various cations (100 μmCaCl2, 100 μm LaCl3, or both) or 5 mm EGTA and 0.3 μm[γ-32P]ATP (Amersham) in a volume of 50 μl, essentially as described previously (13Guerini D. Schröder S. Foletti D. Carafoli E. J. Biol. Chem. 1995; 270: 14643-14650Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and analyzed on 5% Protogel containing the buffer system of Sarkadi et al. (27Sarkadi B. Enyedi A. Földes-Papp Z. Gardos G. J. Biol. Chem. 1986; 261: 9552-9557Abstract Full Text PDF PubMed Google Scholar). 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). 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 (31Hilfiker H. Guerini D. Carafoli E. J. Biol. Chem. 1994; 269: 26178-26183Abstract Full Text PDF PubMed Google Scholar), 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. 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 (28Davis R.E. Singh H. Botka C. Hardwick C. el Meanawy M.A. Villanueva J. J. Biol. Chem. 1994; 269: 20026-20030Abstract Full Text PDF PubMed Google Scholar, 29Davis R.E. Parasitol. Today. 1996; 12: 33-40Abstract Full Text PDF PubMed Scopus (53) Google Scholar). 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) of the predicted 2 in the of all alternative 5′ ends, which are followed by a Thus, the of this gene is apparently by a multipartite which is 4 This analysis also that alternative an amino acid sequence to the of the which is encoded in 2 and in the The of mca-1 cDNA was used in the expression calcium ATPases found in the C. elegans Genome Project in a new of the 5′ of of the gene and the at the 5′ by of the sequence data from the PCR with the genomic sequence in clone primers used for PCR are by reverse transcription-PCR of the alternative first of the mca-1 The which was encoded in (Fig. 2 was by PCR to a FLAG-tagging sequence at the in baculovirus and introduced into the Sf9 cells by A crude membrane preparation from cells a new protein of that monoclonal antibody M2 that the was expressed in Sf9 A crude membrane preparation from cells was to the functional assay Carafoli E. Biochem. Sci. 12: Full Text PDF Scopus Google Scholar) for in the of the PMCA the of the typically be by its in gel (27Sarkadi B. Enyedi A. Földes-Papp Z. Gardos G. J. Biol. Chem. 1986; 261: 9552-9557Abstract Full Text PDF PubMed Google Scholar). The of the assay that a in the be in cells, the expression of the new protein was The was in the (Fig. The an extremely reaction in the of calcium the of reaction in the of the in the of the in the of and calcium to that with pumps of the PMCA family (24Heim R. Iwata T. Zvaritch E. Adamo H.P. Rutishauser B. Strehler E.E. Guerini D. Carafoli E. J. Biol. Chem. 1992; 267: 24476-24484Abstract Full Text PDF PubMed Google Scholar, H. Guerini D. Carafoli E. J. Biol. Chem. 1994; 269: 26178-26183Abstract Full Text PDF PubMed Google Scholar). ATPase of was also by the but it was its was in cells the mca-1 The of this putative which is in the assay on Sf9 cells, is features of the mammalian PMCA such as the and between the the of the sequence of the site P. Zvaritch E. Carafoli E. Biochem. Res. PubMed Scopus Google Scholar) and of the site J. Biol. Chem. Full Text PDF PubMed Google Scholar) as the of the C-terminal that for the of as with those of a SERCA-type pump Strehler E.E. R. R. G. S. Strehler-Page M.-A. P. T. J. Carafoli E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), all to the that the family proteins to the PMCA family ATPases (Fig. 1). Whereas the sequence between mammalian and nematode proteins at the 200 C-terminal amino appears to be the of similarity (Fig. 4 to the of the of the mammalian pumps T. P. J. Enyedi A. Carafoli E. 1990; PubMed Scopus Google Scholar, T. T. F. Carafoli E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In all three nematode this of similarity features an to an with of on one and a of on the (Fig. 4 In a membrane-based the protein human calmodulin at a concentration of 10 nm or This the specific binding of human calmodulin to a which is only in the cells, the mca-1 Moreover, this protein is to have a very similar to the expressed human of used in this out the of an protein (Fig. this is the only one in the of EGTA of human calmodulin to the protein expressed in Sf9 and electroblotted membrane proteins from untransfected cells cells transfected with the mca-1 construct and cells transfected with the construct Sf9 cells were incubated with 10 nm biotinylated human calmodulin and 200 μm The that is only in the of is the protein is The at the show the of the protein marker The of protein in was than that in a is identified by its sequence similarity to in this case, a significant sequence be derived (Fig. 4 which that the family may of the PMCA at a of the nematode other be The genome have in the of proteins with a candidate function from sequence similarity to proteins a genetically identified In this work, database searches have three of EST clones about a similarity to mammalian calcium ATPases of the PMCA The and between the major sequence of the mca-1 protein it in the PMCA family and out the that it to a SERCA-type family or to family found in mammalian In a SERCA-type sequence is encoded on chromosome (cosmid all genes reside on chromosome gene has a of features that are found the of alternative at the the of site A found in the and the of alternative the as with the mammalian alternative site E.E. Strehler-Page M.-A. G. Carafoli E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T.P. Shull G.E. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). A feature protein is however, and is the of the which located between 2 and This the of and acid for Carafoli E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which is in PMCA pumps but in The mammalian PMCA family has been the of studies the after the cloning of the first in the cloned PMCA be expressed in a model system on a cell and of their biochemical be (for a review, see Ref. 3Carafoli E. Garcia Martin E. Guerini D. 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