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Structure of a Plasmodium yoelii gene-encoded protein homologous to the Ca2 -ATPase of rabbit skeletal muscle sarcoplasmic reticulum KENJI MURAKAMI Division of Molecular Biology, Dauchi Pharmaceutical Co. Ltd, Kita-kasai 1-chome, Edogawa-ku, Tokyo 134 KAZUYUKI TANABE* Laboratory of Biology, Osaka Institute of Technology, Ohmiya, Asahi-ku, Osaka 535 and SUEHISA TAKADA Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545, Japan * Author for correspondence Summary A cation-transporting ATPase gene of Plasmodium yoelii was cloned from the parasite genomic library using an oligonucleotide probe derived from a conserved amino acid sequence of the phosphorylation domain of the aspartyl phosphate family of ATPases. The complete nucleotide sequence was determined and it predicts a 126 717 Mr encoded protein composed of 1115 amino acids. Northern blot analysis revealed that the gene is transcribed during the asexual stages of parasite development. The P. yoelii protein contains functional and structural features common to the family of aspartyl phosphate cation-transporting ATPases. The parasite protein shows the highest overall homology in amino acid sequence (42%) to the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Homologies to other aspartyl phosphate cation-transporting ATPases including a plasma membrane Ca2+-ATPase were between 13 and 24 %. The structure predicted from a hydropathy plot also shows 10 transmembrane domains, the number and location of which correlated well with the sarcoplasmic reticulum Ca2+ATPase. On the basis of these results, we conclude that the parasite gene encodes an organellar, but not plasma membrane, Ca2+-ATPase. The P. yoelii protein, furthermore, contains all six amino acid residues in the transmembrane domains that were recently identified as comprising a high-affinity Ca2+-binding site. It follows that organellar Ca2+ATPases of rabbit and Plasmodium conserve functionally important amino acid residues, even though they are remote from each other phylogenetically. Introduction cytosol. Meanwhile, we and other investigators have shown that Plasmodium has a dicyclohexylcarbodiimide (DCCD)-sensitive proton pump at the plasma membrane to generate an inside negative membrane potential (Izumo et al. 1988; Mikkelsen et al. 1982; Mikkelsen et al. 1986; Tanabe, 1983) and utilizes an electrochemical gradient of protons to drive an inward movement of Ca2+ and a sugar from the cytoplasm of infected erythrocytes-(Izumo et al. 1989; Tanabe et al. 1982). These circumstances suggest that Plasmodium exhibits unique mechanisms for transporting ions inside the host cell. In eukaryotes, transmembrane movements of cations are intimately regulated by cation-transporting mem(SR) Ca2++brane ATPases, such as sarcoplasmic reticulum 2+ ATPase, plasma membrane (PM) Ca -ATPase, PM H ATPase, Na+,K+-ATPase and gastric H+,K+-ATPase from a variety of organisms. These ATPases are classified into members of the family of aspartyl phosphate cationtransporting ATPases (or called the P-type or the E1-E2 class of the ATPase) (Pederson and Carafoli, 1987). They Protozoan malaria parasites of the genus Plasmodium undergo repetitive cycles of asexual multiplication in appropriate vertebrate hosts. The parasites grow inside the host's erythrocytes and, after multiplication, destroy the host cell to invade new erythrocytes. Parasitism of Plasmodium presents an intriguing issue with respect to transport of ions and metabolites, because the parasite must adapt itself to both the intracellular and +extracellular 2+ionic environment. Concentrations of Na , K+ and Ca in the erythrocyte cytosol and the plasma of the host differ greatly. Furthermore, as the parasite matures+ intracellularly, the host-cell's cytosolic levels of K decrease greatly and the levels of Na+, concomitantly,+ increase, while the parasite's cytosol maintains a high K and low Na+ level (Lee et al. 1988). This suggests that the parasite has a mechanism by which the cytosolic levels of the alkali cations are maintained constant irrespective of fluctuations of the cation levels in the host erythrocyte Journal of Cell Science 97, 487-495 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 Key words: aspartyl phosphate cation-transporting ATPases, Ca2+-ATPase, endoplasmic reticulum, malaria, parasitic protozoa, Plasmodium, sequence analysis. 487 share common features: a Mr (relative molecular mass) of 100-140 (xlO 3 ), formation of phosphorylated intermediates and high sensitivity to vanadate (Carafoli and Zurini, 1982; Pederson and Carafoli, 1987; Verma et al. 1988). Recent sequence studies by other investigators have revealed that the aspartyl phosphate cation ATPases contain stretches of very conserved amino acid sequences; the site of phosphorylation (James et al. 1987), the FITC (fluorescein isothiocyanate)-binding region (believed to be a part of ATP binding region) (Filoteo et al. 1987) and the FSBA (5'-p-fluorosulfonyl-benzoyladenosine; an analog of ATP)-binding region (Shull and Greeb, 1988; Shull et al. 1985). The most conserved site among them consists of a stretch of seven amino acids, DKTGTLT, in which the aspartate residue serves as the phosphorylation site (Serrano, 1988). We have synthesized an oligonucleotide corresponding to the seven amino acid sequence and have used this oligonucleotide to clone genes for the aspartyl phosphate ATPases of Plasmodium yoelii, a rodent malarial parasite. Here, we describe a clone that encodes a protein homologous to the rabbit SR Ca2+-ATPase and report that the parasite protein conserves functionally important amino acid residues. Materials and methods Parasite The 17XL strain of P. yoelii was maintained by intraperitoneal passages of infected erythrocytes into 6- to 8-week-old female ICR mice (Nippon SLC, Shizuoka, Japan) as described before (Tanabe, 1983). Infected blood collected by cardiac puncture with a heparinized syringe was centrifuged at 800 g for 5min. The sediment was diluted to make a 20 % suspension in Hepes (JV-2hydroxyethyl-piperazine-W -2-ethanesulfonic acid)-buffered saline (HBS: 145 DIM NaCl, 10 mM KC1, lmM MgCl2 and 10 HIM Hepes-NaOH at pH 7.2) and the suspension was passed through a cellulose powder column to remove leukocytes (Richards and Williams, 1973). Eluted erythrocytes were washed in HBS and layered onto a 60 % Percoll gradient for fractionation as described previously (Tanabe, 1983). Cells recovered from the bottom fraction were further fractionated on a 62% Percoll gradient. Cells thus obtained were totally free of leukocytes and platelets. They were then resuspended to 10% in HBS at 37 °C and mixed with an equal volume of 0.04% (w/v) saponin in HBS to lyse erythrocyte membranes for lmin at 37 °C. Erythrocyte-free parasites were sedimented at 10 000 g for 5min at 4°C and washed three times in HBS. Final pellets were used for purifying DNA and RNA. Isolation of DNA and RNA Samples of parasite pellet (0.5 ml) were dissolved in 40 ml of 0.5 M EDTA (ethylenediamine-tetraacetic acid), 0.5% (w/v) Sarkosyl (Sigma Chem., USA) containing 4mg proteinase K (Boehringer, Mannheim, FRG) and incubated at 50°C for 4h. DNA was extracted from the lysed materials with phenol saturated with 50 mM Tris (tris-(hydroxymethyDaminomethane)-HCl (pH8.0) and isolated by ultracentrifugation as described previously (Tanabe et al. 1989). To prepare total RNA, 0.5 ml of the parasite pellet was dissolved in 9 ml of guanidine thiocyanate solution (6 M guanidine thiocyanate, 5mM trisodium citrate, 0.5% (w/v) Sarkosyl and 0.14 M 2-mercaptoethanol) and vortexed vigorously. RNA from the lysed materials was separated by the method of Chirguin et al. (1979). Oligonucleotides A 21-mer oligonucleotide (denoted M15), 5' GATAAAACAGGAACATTAACA 3', corresponding to seven amino acids, DKTGTLT, of a phosphorylation site of the aspartyl phosphate ATPases, was 488 K. Murakami et al. designed by taking into account the A+T-rich codon usage of Plasmodium with a preference for A residues (Weber, 1987) and synthesized from phosphoramidites on an Applied Biosystems DNA Synthesizer. Another 21-mer oligonucleotide (denoted M16), 3' CTATTTTGTCCTTGTAATTGT 5', complementary to M15, was also synthesized. M15 was end-labeled with SOOOCimmoll"1 of [y-32P]ATP (Amersham, UK) and bacteriophage T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan). Southern and Northern hybridizations Three micrograms of P. yoelii DNA were completely digested with EcoRI or HmdIII (Boehringer). The digests were electrophoresed on a 0.8% agarose gel and transferred to a nylon membrane (Hybond N, Amersham) (Weber, 1987). The blots were prehybridized in 6xsodium-EDTA-Tris (SET: lxSET is 0.15M NaCl, 2mM EDTA, 0.03M Tris-HCl at pH8.0), 5xDenhardt's solution (Denhardt, 1966), 0.5% (v/v) Nonidet P40, and lOO^gml"1 salmon sperm DNA for 2h at 45 °C. Hybridizations were conducted under the same conditions for 1 h at 45°C using radiolabeled M15. The blots were washed with 6x standard saline citrate (SSC: lxSSC is 0 . 1 5 M NaCl, 0.015M sodium citrate) for 4 x 30 min at 45 °C, dried, and exposed to Kodak X-Omat AR films at -70°C with intensifying screens. Films were developed in an automated Kodak processor. In some cases, a cloned DNA fragment was radiolabeled with SOOOCimmor1 of [o--32P]dATP (Amersham) by nick translation (Maniatis et al. 1982) using a kit from Boehringer and was used as a probe. Unincorporated label was removed by Sephadex G-50 column filtration (Maniatis et al. 1982). Conditions for prehybridization and hybridization were the same as those for Northern blot analysis (see below). For Northern blot analysis, a sample of P. yoelii RNA (20 fig) was electrophoresed through a 1.0% agarose gel in 2xMops (3(morpholino)propanesulfonic acid) buffer containing 6% (v/v) formaldehyde and transferred to Hybond-N. A mixture of 0.24-9.5 kb (lkb=10 3 bases) RNA fragments from Bethesda Research Laboratories (USA) was used as size markers. The blot was prehybridized in 50% formamide, 5 x standard saline phosphate, EDTA (SSPE; lxSSPE is 0.15M NaCl, 10mM sodium phosphate and 1 mM EDTA at pH 7.4), 5xDenhardt's, 0.5 % (w/v) SDS (sodium dodecyl sulfate), and 20/<gml~1 salmon sperm DNA for 2h at 42 °C. Hybridizations were done under the same conditions using a 1 ;<g nick-translated DNA fragment. Washes were at 42 °C for 2x15 min in 2 x SSPE with 0.1 % (w/v) SDS and for 2x30min in lxSSPE with 0.1% SDS. Construction and screening of DNA libraries One microgram of P. yoelii DNA was digested completely with EcoRI, followed by ligation into the bacteriophage vector AZAP (Stratagene, USA) and packaged in vitro using a kit (Gigapack Gold) from Stratagene (Hohn and Murray, 1987; Sternberg et al. 1977). The library of recombinant bacteriophage was amplified in the LE392 strain of Escherichia coli (Maniatis et al. 1982). Screenings with M15 probe of the library were carried out by plaque hybridization (Benton and Davis, 1977; Maniatis et al. 1978) under the same conditions as described for Southern blot hybridization. DNA inserts of several positive clones were excised and subcloned into the .EcoRI site of Bluescript vector (Stratagene) according to the ZAP excision protocol provided by Stratagene. DNA sequence analysis For sequencing of clone YEL6, the insert was digested with Hindi and subcloned into the Smal/EcoRl sites of pBluescript and ordered deletions along the insert were performed with the help of Exonuclease III and mung bean nuclease (Stratagene). The insert was also subcloned into M13mpl8 and mpl9 (Yanish-Perron et al. 1985), following digestions with a variety of restriction enzymes (AM, Dral, EcoRI, Hindlll and Sau3A). DNA sequence was determined by the dideoxy chain termination method of Sanger et al. (1977) with 35 S- or 32P-labeled dATP using 2'-deoxy-7-deazaGTP instead of dGTP (7-DEAZA sequence kit, Takara Shuzo). Single-stranded DNA obtained from recombinant Bluescript 6 kb vectors and M13 vectors was used a template. Computer analyses of nucleotide and amino acid sequences were performed on an NEC9801 RA5 computer using a SDC-GENETYX program (Genetyx Inc. Tokyo). TAA IHZKDAA OH Southern blot analysis A synthetic oligonucleotide M15 was designed to correspond to a highly conserved stretch of seven amino acids, DKTGTKT, encompassing the phosphorylation domain of the aspartyl phosphate cation-transporting ATPases. To investigate whether M15 could reveal specific DNA fragments, genomic DNA of P. yoelii was digested with EcoRl and was analyzed by the Southern blot technique using the oligonucleotide as a hybridization probe. As shown in Fig. 1, several discrete bands were detectable at 24, 10, 7.2 and 6.0 kb with the strongest signal at 7.2 kb. Isolation and sequence of a P. yoelii clone Screenings with M15 of 2xlO 4 plaques from a genomic library AZAP, yielded 24 positive phage clones with different signal intensities, of which the inserts of four phage clones (denoted YEL3, 5, 6 and 11) were excised and subcloned into pBluescript. They were designated as pYEL3, 5, 6 and 11. The sizes of the inserts of those clones were determined by Southern blot analysis: pYEL3, 5 and 11 had a 7.2 kb insert and pYEL6 a 6.0 kb insert. These inserts corresponded to bands detected by Southern blot analysis of genomic DNA (Fig. 1). To see whether these clones possess genes for cationtransporting ATPases, a partial nucleotide sequence 5' upstream from the M15 sequence of these clones was determined using M16 as a primer (which is complementary to that of M15). pYEL6 has a sequence that encodes a homolog to cation-transporting ATPases (see below). Sequences of pYEL3, 5 and 11 were completely identical but did not appear to encode a cation-transporting ATPase (data not shown). Two base changes (see Fig. 3; A at 1083 and T at 1086) occur between the M15 sequence and the corresponding 21-base sequence in the cloned gene. This could reflect the relatively low signal intensity, obtained for the 6.0 kb band by genomic Southern hybridization. Restriction analysis of pYEL6 revealed the 6.0 kb insert contained a 3.3 kb ifmdIII fragment (Fig. 2). 2.3- I AA SA A AS I Results Fig. 1. Southern blot analysis of P. yoelii genomic DNA digested with EcoBl and probed with the oligonucleotide M15. i AASA AAAA A In f Wf AAAE i DO D Fig. 2. Restriction map of YEL6 encoding a Ca2+-ATPase of P. yoelii. Open column indicates predicted coding regions. Restriction sites are as follows: A, Alul; D, Dral; H, Hindlll; He, Hindi; E, EcoRI; S, Sau3A. The nucleotide sequence of YEL6 and the amino acid sequence deduced from the nucleotide sequence are shown in Fig. 3. Computer analysis demonstrated that there was only one long open reading frame (ORF) starting with ATG codon at residue 1. Upstream from the translation initiation site, there was no ORF with an ATG start codon. The ORF encodes 1115 amino acids with a Mr of 127 xlO 3 and seems to contain two short introns near the C terminus. The reasons for assuming the introns are as follows: (1) predicted introns are short, 115 and 127 bp (base pairs), consistent with other reported introns of malarial parasites, the longest being 438 bp (Weber, 1988); (2) the two sequences of presumed intron/exon boundary, 5'G:GTAAG CTCAG:G3' and 5'G:GTAAT TTTAG:G3', are close to the consensus malaria intron boundary sequence, 5':GTAAG YTAG:3' (Y, pyrimidine) (Weber, 1988); (3) no change in reading frame without assuming introns would result in peptide sequences that are quite different from cation-transporting ATPases; and (4) the second presumed intron has stop codons in any frame. The A+T content in the P. yoelii ATPase gene is high (72 %) and codon usage in the gene is strongly biased in favor of A and T (data not shown), as in the case of P. falciparum, the human malarial parasite (Tanabe et al. 1987; Weber 1988). Comparison of the P. yoelii protein with the aspartyl phosphate cation-transporting ATPases The deduced P. yoelii protein was compared with six aspartyl phosphate ATPases: Ca2+-ATPases from rabbit skeletal muscle sarcoplasmic reticulum (SR) (MacLennan et al. 1985) and from human teratoma PM (Verma et al. 1988), H+-ATPase from Saccharomyces cerevisiae PM (Serrano et al. 1986), H+,K+-ATPase from rat stomach (Shull and Lingrel, 1986), Na + ,K + -ATPase from sheep kidney (Shull et al. 1985), and a cation-transporting ATPase from Leishmania donovani (Meade et al. 1987), a trypanosomatid protozoan parasite. The predicted P. yoelii protein contained all six of the areas previously found to be conserved among the aspartyl phosphate ATPase (Fig. 4). Furthermore, the P. yoelii protein contains conserved peptide sequences for the phophorylation domain, CSDKTGTLT (residues 356 to 364), the FITC-binding region, KGEPE (residues 613 to 617), and the FSBAbinding region, TGDGVNDAPALK (residues 798 to 809) (Fig. 4). In those six conserved regions, the P. yoelii protein showed the greatest amino acid sequence homology (60 %) with the Ca2+-ATPase from rabbit muscle SR (SR Ca 2+ ATPase). The homology between the P. yoelii protein and aspartyl phosphate ATPases other than the SR Ca 2+ ATPase is between 36 and 47 %. Outside of these conserved regions, the P. yoelii protein and the SR Ca2+-ATPase were found to be homologous (Fig. 5). The overall homology Cation-transporting ATPase gene 489 AAATATATGCATATATAACTTG - 2 0 4 ! A«TOTCATAT1TA«TI?rTCATAnCAMTGn«TAmATATGACCCTAnCfflCnCATAAOTATAGMTCAnAnMWTCAGGTATAAGCM -1801 CATAAAmAmAmAAAMOMTAATAmTATATATACAAAATAATATTn^^ -1681 TMnATATCCMTCTAATATCAAAAGGaAAffiAMTTCCTmTATATAmATCATATCATCm™™^ -1561 TrATCACimaTMTAmATCTA^ -1441 ^ -1321 AAATOimAAMTAmTAAMTAMTGAATAGATATATGAGttTAnAAAAAAMMTMTATATATl^ -1201 TTTnCmTACACAATrrTCAmAATTTATATATMnATTAKTATOACTMTOTTTrriTrrrrnOTAffTOAGA -1081 CAMTAGAAAAAAAATAATmATCCAIAOTAAOTAmTTmAAAATMlTGAAAAlTrAGAAATAATATMlTrTCATGATAATrrm -961 CAaATaCGTMCAaAAAAAAAAAAAAAAAAAAAAAAAATOKrrAAAAAAAAATAnAAAATGTAGGATinTnAmAnAAAATnAaCAATATATATTATATACGAATAATGCT -841 OTAnMTMGmmTTmACAAAATTTmATmATTmATCIATGTAAAATAGAIATCCnAlTrATCTATAClTnTm -721 ATATAnATCCmmnATATmATOlTnClTnClTimCCCCCCTTOtOTnOTTnGTTmATMTO -SOI ATACACATATATTAmTMGTAmACCimMGUnATmTAITmATTrAGATATATATATAMGTAMTmnGAGATAAAAAAAGTnAAAAAAATAATAATATAAAAAT -481 TAaCTITGGAmCAAAATAAmTCTCCAAMGAAACMGAAAmMTMCCTATCrAM^ -361 TTTrrrrrTTiTnTrTiTnmcATmTmAA -241 GCATATGCTaTGACTATATGUTATATGTGTATGlWimTGCATCTAMTMG™^ -121 ATATATATGTATAUlOTSTGTmGAATAmCCMTACAmCGATGTATAATAMCTCnWCCATTnCTCAAAAAAATAMTATACAAAATACCMnMTAm -1 a E N I L I T A H I T K V E D V L I A V E V D E I I I G L S E I I E I t l R I I I Q T ATOAAAATATmrnTATGaCATATATACAATCTAGMGATGraTAAGAIM^^ <40> 120 G F H E I E V E E E E G I L E L I L K Q F D D L L V E I L L L A A F V S F A L T WATlTMlWnAGMinTGAGAAAAAAAMGGMTmAGMTrCATAnAMTCMTTTGATGATr^ <80> 240 L L D M B D H E V A l C D F I E P V V I L I l L I l l A A V G V f Q E C S A E E A <120> 360 L E A L K 5 L Q P T K A I V L R D G S I E 1 I D S K I I T V G D I 1 E L S V G TCmAGMGUmAAACMraCMCMCAAMGOimGTGnAAGAGATGGAAMTOTAAA^^ region 1 . H K T P A D A R I V K I F S T S 1 8 A E Q S « L T G E S C S V D B Y V E 8 L DE AATAAAACCCUGaMmOTATAGnAAAATAmCAACMGTAnAAAGUMGCAM . region 2 S L S K C E 1 O . I S B H 1 L F S S T A 1 ¥ A G R C T A V V 1 R 1 G M » T E I C K TanAAAAMnTOAGAnCMnAAAAAAAUTATAnATmCnCTACAGCTATAinWUKTAGATGTACAGOTrrGTMTCAAA^ 1 Q A V 1 E S H H E E T D T <160> 480 <200> 600 <240> 720 P L « l K I D S F G K ( ) L S S I I F I I C ¥ HV f <280> nGTGTACATGTATGG 840 1 1 II F 8 H F S 0 P 1 H E S F L Y G C L Y Y F E 1 S V A L A V A A 1 P E G L P A <320> ITATAnATTnAAMTAAGTGTAGCATTAGaGTTGCTGCAATTCCTGAAGCAmCaGa 9 6 0 region 3 V l T T C L A L G T R R <360> V t t l l A 1 V R 8 L I J S V E T L G C T T V 1 C S 0 8 T A6A^ 1080 G T L T T MQ « T A T ¥ F H 1 F R E S T L K E Y « L C Q R G D T F F F Y E T I I <400> 1200 g D D E » D S F F » K L K E S P » » E S S Y K R K l S l i l i l l D D D D D D T O Y <440> CMATCATGAAAATGATOTTTmTMTmnAAMGMTCACiaMTMTCAATnAGmTAAAAAAAAMTMGTAAAAATATAATAGATGATGATGATGATGATACAGATrAT 1320 E R E P I 1 « « E S H V « T 1 1 S R G S E I 1 D D K I » I! Y 1 Y S D F D Y H F Y <480> GmGAGMCUnMTAMTATGmTCAAATGnAATACAATAATAAGTAGAGCTACTAAMmTAGATMTAAAATAUTAMTATAmAmCMTTTTGAnATCATnTrAT 1440 « C L C » C H E A S 1 L C » ¥ H 1 I B 1 ¥ E T F G D S T E L A L L H F ¥ H N F 1 I I ATGTGTnATGTAATOTAATCMGnAGTATAnATGTAATGnMTMTAAMTOnTAAAAMlTrGMGATAGTACaMTrGGirmACnCAT^ <520> 1560 L P « K T B N N 8 I S « E Y E B I H H 1 T B ( 1 I I S D L I I G G H D S S I Y B B » B 1 S D K K S E P T F P S K C ¥ S A W R » E C T 1 « R 1 I E F T R E R 1 ! L I I S V V AmaGACAAAmTCTCAACCMCAmCCAAGTAMlWWATCreMWAGAMHMTCTACCAnATCAGAAnATTCAAmACTCm region 4 V E N S K« E Y I L r C E C A P E » I I K R C E I T I S t » P I R P L T D S L K CTACAAMTAirrAAAMTGAATATAlTnATAlTCTAAAGGTGCACCAGAAAATAmTmTAGATCTAMTAmTATOTCAAAmTCATATACaCCAnAACAGAnCAn <600> 1800 <640> 1920 » E 1 L H 5 I l l H ) I G K R A L R T L S F A Y I ! l ! ¥ K S » D l l ( I l i « S E D Y Y <680> AATCAAATTTTmTAAMTAAAAMTATGGGAAAUGAGCmAAGAACmMGTTrrGCATATAAAAAAGnAAAiaMTCATAnMTATAAAAAATrCIIMGATOTOTAM 2040 region 5 L E H D L 1 Y I G G L G I I D P P R i Y V G K A l S L C H L A C l R V F » l T G <720> mGMCATGATTTMATATATAGCAGGATTGKMmTTGATCaCMCGAMATATGTAGGAAUGCTAraGmATCTCATTraCAGGTA^ 2160 8 II I D T A 8 A 1 A K E 1 N I L I H D D T D 8 Y S C C F K G R E F E D I P L E 8 GATAATATAGATACTCaAMGCTATItamfaMnMUTATrGAATUTMTaTACAMTAMTATAGTlWTGTTrrAATaaan^ <760> 2280 Q K Y 1 L K N J Q <S00> O l V F C B T E P t H K H H I V I I L K D L C E T V A I I T G D region 6 G V K 0 A P A L K S A D 1 G I A H G I H G T 8 V A I I L A D D H F II T <840> ITTnGGCTGATGATAATTTrAACACC 2520 1 V E A 1 R E G R C I Y » H » S A F 1 R Y L I S S 1 I I G E ¥ A S 1 F I T A 1 L G ATOTTGMGCTAnAAAGMGffrCGATGTATATATMCMMTGmGCTmATACGATATClTATMGTACTAATATTOAGAAGTaCnCGATTnamCTC <880> 2640 P D S L A P V O . L L » V H L ¥ T D G L P A T A L G ! | F » P P E » D V « B C 8 P R H R mCTacnCTCAmnMcccca^^ H D N L 1 K G L T I L R Y 1 V 1 G T Y V G I A T V S 1 F 1 Y W Y M F Y P D M D I I AAAraMTTTMTmCWKCTMCCCTCCTAAGATAUTACTMTAreMmATGTTGGMTAGCTACAirrGTCGATAmATATATTOrrACAT^^ H T L I » F Y ( ! L S H Y » Q C l ! T f S I I F ! l ¥ I I K V Y D ) I S E D L C S Y F S A G <922> 2880 <962> 3000 <1002> 3120 <1005> K V I ! AAAGGTCMGGTAAmCTACTGCCGCnaUMTCTATATCTHTaTATGaTATCATGTnATCaMnATATGCanmAGmCCCTAACGATGTCMCACCTAmGTA^ 3240 l A S T L S L S V L V L I E K F N A L S A L S E I H S L F V L P P I R I i AinmttTTrmAGiK^CTAanATunATcrinTnAcrmMTOMimcMTCimAMTmn^^ <1040> 3360 H Y L V L A T 1 G S L F L H C L 1 I Y F P P L A G 1 F G V V P L T L H D W F L V <1080> ATATGTAmAGTACmCAACMTOWCTCTCTAmOTCAmmMTMTATAm 3480 F L W S F P V l I l E l l S F Y A E E Q L H K E L G Y G I l K L B T g TmATO^AAAAAAMaAMTAAGMGirr^ AMTACTCAATATUCAGACATGCAGACAGAUTAUGAMGACATAUGACAGAaCACMTGAGAGAAnCGATATCAAGCTr <111S> 3600 3773 Fig. 3. Nucleotide sequence of P. yoelu ATPase gene (YEL6) and deduced amino acid sequence. Nucleotides and amino acids are numbered in the right margin. Amino acid sequences underlined are those conserved among aspartyl phosphate cation-transporting ATPases, region 1-6. Asterisks indicate the domain of phosphorylation from which M15 was designed; and vertical bars indicate the boundaries of predicted introns. IDSKYLTVGDt IEL:SVGNKTPAUAHIVKIFSTSIKAECJSKCLTGB'SCSVDKY fKAKDIVPGDfVBIAVGDKVPAT)ITiLTSrKSTTLRVDQSaTGE'SVSVIKH IPVADITVGDIAQVKYGDLLPABG-IL-IQGNDLKIDESSLTGBSDHVKKS [PANEVVPSDfLQLEDOTVlPTbGfilVTEDCF-LQIDflSAIIGESLAVDKH .INADQLVVGDLVEMKGGDRVPADIR1LSAQGC--KVDNSSLTGBSEPQTRS JNAEEVVVGfDLVEVKGGDRIPADLR! ISANGC- -KVDN^SLTGESEPQTRS IDAAVLVPODLVKLASGSAVPASCSINEGVID-••VDEAALfGBSLPVTMG PYEL6 SRCAS PMCAT PMASC HKARS NKASK LdATP 144 140 205 191 189 178 165 PYEL6 SRCAS PMCAT PMASC HKARS NKASK LdATP 214 LFSSTAIVAGRCTAVVIKIGMNTBJGNIQHAVIESNHEBTD 208 LFSGTNIAAGKAMGVWATGVNTEIGKIRDEMVATEQERTP 260 iLSGTHVRECSGRMyYTAVGVNSQTOllFTLLGAGGEEBEK 2 4 5 TF.SSSTVKRGEGFM:VVTATqDNTFVQRAAALV.NKAAGGQGH 252 APFSTMCLEGTAQGLVVSTGDRTHGRIASLASGVENEKTP 2 4 1 AFFSTNCVEGTARGIVVYTGDRTVMGRIATLASGLEGGQTP 217 PKMGSNVVRGEVEGTVQYTGSLTFFGKTAALLQSVESDLGN 194 190 253 240 237 226 212 254 248 300 285 292 281 257 PHOSPHORYLATIDN PYEL6 SRCAS PMCAT PMASC HKARS NKASK LdATP 307 300 424 327 334 323 300 PYEL6 SRCAS PMCAT PMASC HKARS NKASA LdATP 610 LYCKGAPENTlNRCKYYMOTDIRPLTDSLKNEtL 644 511 MFVRCAPEGVtDKCTHIRVGSTKVPMTAGVKQKlM 545 598 IFSKGASEI-ILKKCFKILSANGEAKVFRPRDRDDI 632 471 VCVKGAPLSALKTVEEDHPIPEDVHENYENKVAEL 505 514 iVMKGAPERVLERCSSILIKGQELPLDEQWREAFQ 548 503 :' .v.vH; I'..;- :r~MLlHGKEQPLDEELKDAFQ 537 442 : ..-.,;\.-...1 .,'-'. •-'. QDEIKDEVVD11DSLAARG 476 PYEL6 SRCAS PMCAT PMASC HKARS NKASK LdATP 695 600 684 534 602 591 501 iJRPRKYVGKAtSLGHLAGtHVFMITGDJfIDtAK DRPBIBVASSVKLCRQAGIBVI»[TGDNKGTAV DPVRPEVPDAIKKCQRAGITVRMVTG1MNTAR DPPRDDTAQTVSEARHLGLRVKMLTGDAVGIAK DPPRATVPDAVLKCRTAGfRVlMVTGDHPITAK DPPRAAVPDAVGKCRSAGIKVIMVTGDHPITAK DPPRPDTKDTJRRSKEYOVD\?KMITGDHLLIAK PYEL6 SRCAS PMCAT PMASC HKARS NKASK LdATP 770 672 762 604 696 685 575 QIVFCRTEPK8K KNIVKILKDLGBTVAMTGDGVSDAPALKSADIGIAMGINGTQYAKEASD ARCPAfiVEPSBK SKI?EFLQSFDBITAMTGDCVIIMIPALKKAEIGt^UQS• CTAVAKTASE LRVLARSSHDKHTLVKGI iDSTVSDQRQVVAVTGDGTHBGPALKKABVGFAMGlAGTDVAteASD ADGPAEVFPQHK YRVVEILQNRGYLVAMTGDGVNDAPSLKKADTGIAVEGA-TDAARSAAD EMVFARTSPQQK LVIVESCQRLGAIVAVTGDGVMDSPALKKAB[GVAMGIAGSDAAKNAAD EIVFARTSPQQK LIIVEGCQRQGAIVAVTGDGVSDSPALKKAD1GVAMG1AGSDVSKQAAD VGGPAQVFPEHK FMIYETLRQRGYTCAMTGBGVIMF'ALKRADVGIAVHDA-TDAARAAAD VAIAVAA IPEGLPAVITTCULGTRRMVKKNAIVRK LQSVBTLGCTTVICSDKTGTLTTN VALAVAAlPEGLpA.VlTTCLALGfRgMAKKSA!VRSLPS.VETLGCTSV}CSDKTGTLtTN VTVLVVAVP.EGLPLAVTlSUYSVKKMMKDliINLVRHLDACElMGNAtAffc$D](TGtLTMJl LGIT11GVPVGLPAVVTTTMAVGAAYLAKKQAi?QKLSAIBSLAGVE1LCSDKTGTLTKN MAIVVAYVPEGLLATVTVCLSLTAKRLASKNCVVKNLEAVETLGSTSVfCSBKTGTLTQN IGIIVANVpEGLLATVTVCLTLTAKSMARKNCLVKNLEAVETLGSTSTlCS&KTGTLtQN VVVL.VVSiBIALEIVVTTTLAVGSKHLSKHKnVTKLSAIEMMSGVNMLCSDKTGTLTLN 366 359 483 386 393 382 359 FITC 727 632 716 566 634 623 533 FSBA between the two proteins is 42 %. The P. yoelii protein is more divergent from other cation-transporting ATPases; the overall homology is 24% for Na+,K+-ATPase, 23 % for H+,K+-ATPase, 16% for yeast H+-ATPase and 13% for 830 731 827 663 756 745 634 Fig. 4. Amino acid homology between the P. yoelii ATPase and six aspartyl phosphate cationtransporting ATPases. Gaps were introduced to maximize homology. Identical residues are shaded. The domains of phosphorylation, FITCbinding, and FSBA-binding are indicated by continuous lines above the sequences. Designation of sequences is as follows: PYEL6, the P. yoelii ATPase; SRCAS, the Ca 2+ ATPase of rabbit slow-twitch skeletal muscle sarcoplasmic reticulum (MacLennan et al. 1985); PMCAT, the Ca2+-ATPase of human teratoma plasma membrane (Verma et al. 1988); PMASC, the H+-ATPase of Saccharomyces cerevisiae plasma membrane (Serrano et al. 1986); HKARS, the H+,K+-ATPase of rat stomach (Shull and Lingrel, 1986); NKASK, the Na+,K+-ATPase of sheep kidney (Shull et al. 1985); LdATP, a cation-transporting ATPase of Leishmania donouani (Meade et al. 1987). Ca2+-ATPase from the human teratoma PM (PM Ca 2+ ATPase) and the Leishmania donovani ATPase. Hydropathy analysis predicted 10 hydrophobic transmembrane domains; four at the amino terminus and six at Cation-transporting ATPase gene 491 the carboxyl terminus (Fig. 6). The N-terminal and Cterminal putative transmembrane domains were separated by a non-hydrophobic central part of 535 residues in length. All six aspartyl phosphate ATPases exhibited four transmembrane domains at the N terminus, followed by a non-hydrophobic central part. Their C-terminal portion contained four to six transmembrane domains: the exact number and which of the hydrophobic domains actually span the membrane are still the subjects of active research (Shull et al. 1985; Verma et al. 1988). It is clear from Fig. 6 that the number and location of the 10 putative transmembrane domains of the P. yoelii protein coincide well with those of the SR Ca2+-ATPase but not to other ATPases. The SR Ca2+-ATPase contains amphipathic stalk sectors MENILKYAHIYNVEDVLRAVKVDENRGLSENEIRKRIMQYGFNELEVEKKKGILELILNQFDDLLVKILLLAAFVSFALTLLDMKDNEVA 90 MEN-•••AHTKTVEEVLGHFGVNESTGLSLEQVKKLKERWGSNELPAEEGKTLLELVIEQFEDLLVRILLLAACISFVLAWFEEG--EET 8 4 SI Ml LCDFIEPVVILMILILNAAVGVWQECNAEKSLEALKQLQPTKAKVLRDGKWEI••1DSKYLTVGDIi ELSVGNKTPADARIVKIFSTSIK 1 7 8 ITAFVEPFV1LL1LVANA1VGVWQERNAENAIEALKEYEPEMGKVYRQDRKSVQRIKAKDIVPGDIVEIAVGDKVPADIRLTSIKSTTLR 1 7 4 M2 S2 AEQSMLTGESCSVDKYVEKLDESLKNCEIQLKKNILFSSTAIVAGRCTAVV1K1GMNTEIGNIQHAVIESNNEETDTPLQ1KIDSFGKQL 2 6 8 VDQSILTGESVSVIKHTDPVPDP- -RAVNQDKKNMLFSGTNIAAGKAMGVVVATGVNTEIGKiRDEMVATEQERJ.-.-PLQQKLDEFGEQL 2 6 0 S3 SK11F11CVHVWIiNFKHFSDPIHE•SFLYGCLYYFKiSVALAVAAIPEGLPAVITTCLALGTRRMVKKNAIVRKLQSVETLGCTTVICS 3 5 7 SKVISLIClAVW11NIGHFNDPVHGGSW1RGA1YYFK1AVALAVAA t PEGLPAV1TTCLALGTRRMAKKNA1VRSLPSVETLGCTSV1CS 3 5 0 M3 M4 S4 DKTGTLTTNQMTATVFHIFRESNTLKEYQLCQRGDTFFFYETNQDDENDSFFNKLKESPNNESSYKKKISKNIIDDDDDDTDYEREPLIN 4 4 7 DKTGTLTTNQMS VCRMF1LDKVDGETCSLNE 3 8 1 MKSNVNT11SRGSK11 DDK INKYIYSDFDYHFYMCLCNCNEASILCNVNNKIVKTFGDSTELALLHFVHNFNILPNNTKNNKISMEYEKI 5 3 7 FTITGSTYAP1GEVHKDDKPVKCHQTDGLVELATICALCNDSALDYNEAKGVYEKVGEATETALTCLVEKMNVFDTELKGL NNITKQNSDLNGGHDSSTYKKNKISDKKSEPTFPSKCVSAWRNECTIMRll-EFTRERKLMSVVVENSKNEYiLYC 462 KGAPENIINRC 6 2 3 SKIERANACNSVIKQLMKKEFTLEFSRDRKSMSVYCTPNKPSRTSMSKMFVKGAPEGV i DRC 5 2 4 KYYMSKNDIRPLTDSLKNEILNKIKN--MGKRALRTLSFA-•AYKVKSNDINIKNSEDYYKLEHDLIYIGGLG11DPPRKYVGKA!SLCH 7 0 9 THIRVGSTKVPMTAGVKQKIMSVIREWGSGSDTLRCLALATHDNPLRREENHLKDSANF1KYETNLTFVGCVGMLDPPRIEVASSVKLCR 6 1 4 LAG 1RVFMITGDNIDTAKAIAKEINILNHDDTDKYSCCFNGREFEDLPLEKQKYILKNYQQIVFCRTEPKHKKN1VKILKDLGETVAMTG 7 9 9 QAGIRVIMiTGDNKGTAVAICRRIGIFGQEE-DVTAKAFTGREFDELNPSAQR-'DACLNARCFARVEPSHKSKIVEFLQSFDEITAMTG 7 0 1 DGVNDAPALKSADIGIAMGINGTQVAKEASD11LADDNFNTIVEAIKEGRCIYNNMKAFIRYLISSNIGEVASIFITAILGIPDSLAPVQ 8 8 9 DGVNDAPALKKAE1GIAMGS•GTAVAKTASEMVLADDNFSTIVAAVEEGRA1YNNMKQF1RYL1SSNVGEVVC1FLTAALGFPEAL1PVQ 7 9 0 S5 M5 LLWVNLVTDGLPATALGFNPPEHDVMKCKPRHRNDNL1NGLTLLRY i VIGTYVGIATVSIF1YWYMFYPDMDNHTL1NFYQLSHYNQCKT 9 7 9 LLWVNLVTDGLPATALGFNPPDLD1MNKPPRNPKEPL1SGWLFFRYLA1GCYVGAATVGAAAWWF1•••AADGGPRVSFYQLSHFLQCK• 8 7 6 M6 M7 WSNFNVNKVYDMSEDLCSYFSAGKVKASTLSLSVLVLIEMFNALNALSEYNSLFVLPPWRNMYLVLATIGSLFLHCLIiYFPPLAGIFGV 1 0 6 9 ••••EDNPDFE-GVD-CA1F--ESPYPMTMALSVLVTIEMCNALNSLSENQSLLRMPPWENIWLVGS1CLSMSLHFL1LYVEPLPLIFQ1 9 5 8 M8 M9 VPLTLHDWFLVFUSFPV111DE11KFYAKKQLNKELGYGQKLKTQ 1115 TPLNVTQWLMVLK1SLPVILMDETLKFVARNYLEPA1LE M10 997 Fig. 5. Comparison of amino acid homology between the P. yoelii ATPase (top line) and the Ca2+-ATPase of rabbit slow-twitch skeletal muscle sarcoplasmic reticulum (bottom line). Residues conserved between the two proteins are indicated by double dots. Gaps were introduced to maximize homology. Ten transmembrane domains, Ml to MIO, and five stalk sectors, SI to S5, of the Ca -ATPase are indicated by continuous and broken underlines, respectively. 492 K. Murakami et al. PYEL6 -3.S 3.0 SRCAS Q.S -3.0 3.a WtyAifiyb^^ PMCAT -3.Q 3.a PMASC 1221 -3. a 3.0 HKARS 1221 -3.S 3.0 -s.e r^WV NKASA w*y 1221 3.0 e.a A LdATP V \A> -3.0 (Brandl et al. 1986; MacLennan et al. 1985), adjacent to each of the five transmembrane domains, designated as Ml to M5 (Fig. 5), that are presumed to form a gate for Ca 2+ . Possible equivalent stalk sectors also occur in the P. yoelii protein at predicted corresponding regions (Fig. 5). Conservation of amino acid sequence between the SR Ca2+-ATPase and the P. yoelii protein is high in the five stalk sectors (65%) and in the ten transmembrane domains (63 %). Homology between the P. yoelii protein and the SR Ca2+-ATPase in the non-hydrophobic central part is rather low although nearly perfect conservation occurs in the phosphorylation domain, the FITC-binding region and the FSBA-binding region (Fig. 5). This is not surprising because the non-hydrophobic central part of the aspartyl phosphate ATPase family varies strikingly in both size and sequence (see Fig. 6) (Shull and Greeb, 1988; Verma et al. 1988). Furthermore, amino acid sequences in this central part are highly conserved between the P. yoelii protein and a homologous protein of P. falciparum, the human malarial parasite (Kimura and Tanabe, unpublished data). It is of interest to note that the central part of the P. yoelii protein contains two stretches of hydrophilic segments that are rich in charged residues: lysine, arginine, aspartic acid and glutamic acid. Northern blot analysis To conduct Northern blot hybridization a 3.3 kb Hindlll DNA fragment isolated from pYEL6 was used as a probe. The probe, which encompasses three fourths of the 5' end of the coding region, detected only a single band at 4.4 kb under low- and high-stringency washing conditions (Fig. 7B). In contrast, a 7.2 kb £coRI fragment of YEL3 did not react with the parasite RNA (Fig. 7C). A Fig. 6. Hydropathy plots for aspartyl phosphate cationtransporting ATPase. The plots were made using a window size of 20 and the Kyte-Doolittle algorithm. The putative 10 transmembrane domains of the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SRCAS) proposed by Brandl et al. (1986) are shown filled for comparison. See Fig. 5 for abbreviation of ATPases. B kb 9.57.54.42.41.4- 0.24- Fig. 7. Northern blot analysis of ATPase RNA expressed in P. yoelii. Blots were probed either with a 3.3 kb (lkb=10 3 base pairs) Hindlll DNA fragment of YEL6 (B) or with a 7.2 kb £coRI fragment of YEL3 (C). (A) RNA size markers. Discussion The present results indicate that P. yoelii has a cationtransporting ATPase gene that is expressed at the blood stage in parasite development. The amino acid sequence coded by a genomic clone contains stretches of sequences that are shared by members of the family of aspartyl phosphate cation-transporting ATPases (Hager et al. 1986; Shull and Greeb, 1988; Serrano, 1988); i.e. the phosphorylation domain, the FITC-binding region and the FSBACation-transporting ATPase gene 493 Table 1. Conservation of Ca -binding amino acids among aspartyl phosphate cation-transporting ATPases Amino acid residue* ATPase species M4t M5 M6 M6 M6 M8 P. yoelu ATPase Rabbit SR Ca2+-ATPase Human PM Ca2+-ATPase Yeast H+-ATPase Rat stomach H+,K+-ATPase Sheep kidney Na+,K+-ATPase L. donovani ATPase E316 E309 E433 V336 E343 E327 1309 E869 E771 A866 E703 E795 E779 V674 N894 N796 N891 A726 E820 D804 T800 T897 T799 M894 A729 T823 T807 N803 D898 D800 D895 D730 D824 D808 D804 E1018 E908 N977 E803 E936 V920 E800 * High-affinity Ca2+-binding site identified by Clarke et al. (1989) are aligned according to the sequence alignment reported by Clarke et al. (1989) and Meade et al. (1987), with a minor modification, i.e. positions of residues are from the SR Ca2+-ATPase of rabbit slow-twitch muscle, instead of fast-twitch muscle. t Transmembrane domain number proposed by Brandl et al. (1986). binding region. Among the six aspartyl phosphate ATPases compared, a cation-transporting ATPase of P. yoelii shows the highest amino acid sequence homology to the SR Ca2+-ATPase of rabbit skeletal muscle. We consider the P. yoelii protein to be a Ca2+-ATPase for the following reasons: (1) the highest overall homology to the SR Ca2+-ATPase; (2) the highest sequence similarity to the SR Ca2+-ATPase in six conserved regions of aspartyl phosphate ATPases (Fig. 4); (3) a close resemblance to the hydropathy profile of the SR Ca2+-ATPase (Fig. 6); and (4) a high sequence conservation in 10 putative transmembrane domains. The last point deserves attention because low homology is found for those domains in the aspartyl phosphate cation-transporting ATP-ases (Shull and Greeb, 1988; Verma et al. 1988). What should be emphasized in this context is that the P. yoelii protein possesses all the amino acid residues in the transmembrane sequences, M4, M5, M6 and M8 (Table 1), which have been recently identified to be the site of high-affinity Ca 2+ -binding in the SR Ca2+-ATPase (Clarke et al. 1989a,6). Thus, it follows that, as far as transport of Ca 2+ in eukaryotic organelles is concerned, the functionally important domains and amino acid residues of Ca2+-ATPase are highly conserved, even though the organisms are far distant evolutionarily. Since antibodies against purified SR Ca2+-ATPase crossreact with a protein in the endoplasmic reticulum of liver cells (Damiani et al. 1988), it has been considered that nonmuscle cells contain and express genes for organellar Ca2+-ATPases (Heilman et al. 1984). Recent sequence studies have revealed the presence of at least three isoforms of SR Ca2+-ATPase in mammalian muscle and non-muscle cells (Burk et al. 1989). It is important to note that they are divergent from PM (PM) Ca2+-ATPase (Shull and Greeb, 1988; Verma et al. 1988) and do not contain a calmodulin binding region. A Ca2+-ATPase of P. yoelii shows almost equal amino acid sequence similarity to these three isoforms of mammalian organellar Ca 2+ ATPase (data not shown). In non-muscle cells, an organellar Ca2+-ATPase is localized at membranes of either the endoplasmic reticulum or the calciosome, an organelle proposed recently as a cytoplasmic Ca 2+ pool (Volpe et al. 1988). Burgoyne et al. (1989) have suggested that, using monoclonal antibodies against the SR Ca 2+ ATPase of rabbit skeletal muscle, the endoplasmic reticulum and calciosomes of bovine adrenal chromaffin cells have different Ca2+-ATPase-like proteins: one with a Mr of 100 xlO 3 that is diffusely distributed in the cytoplasm (probably calciosomes) and the other with a MT of 140 x 103 that is restricted to a region in close proximity to the nucleus (probably endoplasmic reticulum). Both organelles are thought to act in regulating cytoplasmic levels of 494 K. Murakami et al. Ca 2+ by operating their membrane Ca2+-ATPases in nonmuscle cells. Since Plasmodium is a eukaryotic organism, there is no reason to believe that the parasite has a unique type of regulation of cytoplasmic Ca levels. Hence, data strongly suggest that the P. yoelii protein is the same as parasite organellar Ca2+-ATPase. The P. yoelii Ca2+-ATPase and SR Ca2+-ATPase differ greatly in sequences of 220 amino acid residues starting shortly after the site of phosphorylation. This region of sequence shows very low homology among the aspartyl phosphate cation-transporting ATPases, even for isoforms of SR Ca2+-ATPases (Burk et al. 1989). A recent finding (James et al. 1989) that a peptide sequence that binds to phospholamban (PLN) occurs in the C-terminal phosphorylation domain is of interest. PLN is a protein that controls the activity of the SR Ca2+-ATPase of a rabbit skeletal muscle (Tada and Katz, 1982). The P. yoelii Ca 2+ ATPase does not contain the PLN-binding site but, instead, has two regions of peptide sequence rich in charged residues (at positions 377 to 443 and 526 to 567). The PM Ca2+-ATPases of rat brain and human teratoma possess calmodulin-binding sequences rich in charged residues at the C terminus (Shull and Greeb, 1988; Verma et al. 1988). Thus, it may be that the activity of P. yoelii Ca2+-ATPase is controlled by an unknown regulatory protein. In eukaryotic cells, cytoplasmic Ca 2+ is sequestered into cell organelles, the endoplasmic reticulum, the calciosome and the mitochondrion. Ca 2+ accumulates in the mitochondrion by a transport process dependent on a high potential difference (inside negative) across the inner membrane of the organelle. We have previously noted that Ca 2+ transport processes in the parasite is less sensitive to 1 mM KCN and 10 HIM NaN3, inhibitors of mitochondrial electron transport (Tanabe et al. 1982). This implies that the Plasmodium mitochondrion is not actively involved in the regulation of cytoplasmic levels of Ca 2+ . The asexual forms of Plasmodium spend much of the cell cycle inside the host erythrocyte, in which the cytoplasmic levels of Ca 2+ are extremely low. This is in contrast to the high levels of Ca 2+ found in extracellular fluids of most eukaryotic cells. We have previously shown that the influx of Ca 2+ is increased in erythrocytes infected with P. chabaudi, a rodent malarial parasite, and that Ca 2+ is almost exclusively localized in the parasite compartment but not in the erythrocyte cytoplasm (Tanabe et al. 1982). It is, however, quite unlikely that Ca 2+ levels increase evenly in the parasite cytoplasm. Instead, we consider the cation to be sequestered in intracellular Ca 2+ pools (the endoplasmic reticulum or calciosomes). Therefore, regulation of the cytoplasmic concentration of Ca 2+ by an organellar Ca2+-ATPase is of vital importance. The parasite Ca2+-ATPase would therefore be able to participate intimately in the fine-tuning of Ca 2+ and Ca 2+ dependent metabolic processes in the parasite cytoplasm. We thank Dr M. Furusawa for encouragement during the work. This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Science, Education and Culture (nos 01570219 and 02670172). MANIATIS, R., FRITSCH, E F. AND SAMBROOK, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. MANIATIS, T., HARDISON, R. C , LACY, E., LANER, J., O'CONNELL, C, QUON, D., SIM, G. K. AND EFSTRATIADIS, A. (1978). The isolation of structural genes from libraries of eucaryotic DNA. Cell 15, 687-701. MEADE, J. C , SHAW, J., LEMASTER, S., GALLAGHER, G. AND STRINGER, J. R (1987). Structure and expression of a tandem gene pair in Leishmania donovani that encodes a protein structurally homologous to eucaryotic cation-transporting ATPases Molec. cell. Biol 7, 3937-3946. MIKKELSEN, R. B., TANABE, K. AND WALLACH, D F. H. (1982). References BENTON, W. D. AND DAVIS, R. W (1977). Screening of Agt recombinant clones by hybridization to single plaque in situ. Science 196, 180-182. BRANDL, C. J., GREEN, N. M., KORCZAK, B. AND MACLENNAN, D. H 2+ (1986). Two Ca -ATPase genes: Homologies and mechanistic implications of deduced amino acid sequences. Cell 44, 597-607. BURGOYNE, R. D., CHEEK, T. R., MORGAN, A., O'SULUVAN, A. J., MORETON, R. B., BERRIDGE, M. J., MATA, A. M., COLYER, J., LEE, A. G. AND EAST, J. M. (1989). Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells. Nature 342, 72—74. Membrane potential of P/asmodium-infected erythrocytes. J. Cell Biol 93, 685-689. MIKKELSEN, R. B., WALLACH, D. F. H , DOREN, E. V. AND NILLNI, E. A. (1986). Membrane potential of erythrocytic stages of Plasmodium chabaudi free of the host cell membrane. Molec. Biochem. Parasit 21, 83-92. PEDERSON, P. L. AND CARAFOLI, E. (1987). Ion motive ATPases. I. Ubiquity, properties, and significance to cell function. Trends biochem. Sci 12, 146-150. RICHARDS, W. H G AND WILLIAMS, S. G. (1973). The removal of cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2+ pump J. biol Chem. 264, 18 561-18 568. CARAFOLI, E AND ZURINI, M (1982) The Ca2+-pumping ATPase of plasma membranes. Biochim. biophys. Ada 683, 279-301. leucocytes from malaria infected blood Ann. trop. Med. Parasit. 67, 249-250. RlGBY, P. W. J., DlECKMANN, M., RHODES, C. AND BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. molec. Biol 113, 237-251. CHIRGUIN, J. M., PRYZYBYLA, A. E., MACDONALD, R. J. AND RUTTER, W. SANGER, F., NICKLEN, S. AND COULSON, A. R. (1977). DNA sequencing J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. with chain terminating inhibitors. Proc. natn Acad. Sci. U.S.A. 74, 5463-5467. BURK, S. E., LYTTON, J., MACLENNAN, D. H. AND SHULL, G. E. (1989). CLARK, D. M., LOO, T. W., INESI, G. AND MACLENNAN, D. H. (1989a). Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature 339, 476-478. CLARKE, D. M., MARUYAMA, K., LOO, T. W., LEBERER, E , INESI, G. AND MACLENNAN, D. H. (19896). Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum. J. biol. Chem. 264, 11246-11251. DAMIANI, E., SPAMER, C, HEILMANN, C, SALVATORI, S. AND MARGRETH, A. (1988) Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca2+-ATPase and calsequestrin. J. biol. Chem. 263, 340-343. DENHARDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. biophys. Res. Commun. 23, 641-646. FILOTEO, A G., GORSKI, J P AND PENNISTON, J. T (1987). The ATP- binding site of the erythrocyte membrane Ca 262, 6526-6530. 2+ pump. J. biol. Chem. HAGER, K. M., MANDALA, S. M., DAVENPORT, J. W., SPEICHER, D. W., BENZ, E. J. JR. AND SLAYMAN, C. W. (1986). Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences. Proc. natn. Acad. Sci. U.S.A. 83, 7693-7697. HEILMANN, C, SPAMER, C. AND GEROK, W. (1984). The calcium pump in rat liver endoplasmic reticulum. J. biol. Chem. 259, 11139-11144. HOHN, B. AND MURRAY, K. (1985). Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. natn. Acad. Sci. U S.A 74, 3259-3263. IZUMO, A., TANABE, K. AND KATO, M. (1988). The plasma membrane and mitochondrial membrane potentials of Plasmodium yoelu. Comp Biochem. Physwl. 91B, 735-739. IZUMO, A., TANABE, K., KATO, M., DOI, S., MAEKAWA, K. AND TAKADA, S. (1989) Transport processes of 2-deoxy-D-glucose in erythrocytes infected with Plasmodium yoelu, a rodent malaria parasite. Parasitology 98, 371-379. JAMES, P., INUI, M , TADA, M., CHIESI, M. AND CARAFOLI, E (1989). Nature and site of phospholamban regulation of the Ca 2+ pump of sarcoplasmic reticulum. Nature 342, 90-92. JAMES, P., ZVARITCH, E. I., SHAKHPATONOV, M. I., PENNISTON, J. T. AND CARAFOLI, E. (1987). The amino acid sequence of the phosphorylation domain of the erythrocyte Ca2+ ATPase. Biochem. biophys. Res. Commun. 149, 7-12. LEE, P., YE, Z., VAN DYKE, K AND KIRK, R. G. (1988). X-ray microanalysis of Plasmodium falciparum and infected red blood cells: effects of qinghaosu and chloroquine on potassium, sodium, and phosphorous composition. Am. J. trop. Med. Hyg. 39, 157-165. MACLENNAN, D. H., BRANDL, C. J., KORCZAK, B. AND GREEN, N. M. (1985). Amino-acid sequence of a Ca2++Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence Nature 316. 696-700 SERRANO, R., KIELLAND-BRANDT, M C AND FINK. G. R. (1986). Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K + ), K + and Ca2+-ATPases. Nature 319, 689-693. SERRANO, R. (1988). Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochim. biophys. Acta 947, 1-28 SHULL, G. E. AND GREEB, J. (1988). Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. J. biol. Chem. 263, 8646-8657. SHULL, G. E. AND LINGREL, J. B. (1986). Molecular cloning of the rat stomach (H++K+)-ATPase. J. biol Chem. 261, 16 788-16 791. SHULL, G E., SCHWARTZ, A. AND LINGREL, J. B. (1985). Amino Acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA. Nature 316, 691-695. STERNBERG, N., TIEMEIER, D. AND ENQUIST, L. (1977). In vitro packaging of a lambda vector containing EcoRI fragments of Escherwhia coli and phage PI. Gene 1, 255-280. TADA, M. AND KATZ, A. M. (1982). Phosphorylation of the sarcoplasmic reticulum and sarcolemma A. Rev. Physwl. 44, 401-423. TANABE, K. (1983). Staining of Plasmodium ,voe/u-infected mouse erythrocytes with the fluorescent dye rhodamine 123. J. Protozool. 30, 707-710 TANABE, K., MACKAY, M.. GOMAN. M. AND SCAIFE, J. G. (1987). Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum. J. molec. Biol. 195, 273-287. TANABE, K., MIKKELSEN, R. B. AND WALLACH, D F. H. (1982). Calcium transport of Plasmodium chabaudi-mfccted erythrocytes. J. Cell Biol. 93, 680-684. TANABE, K., MURAKAMI, K. AND DOI, S. (1989). Plasmodium falciparum: dimorphism of the pl90 alleles Expl Parasit 68, 186-189. VERMA, A. K , FILOTEO, A. G.. STANFORD, D. R., WIEBEN, E. D., PENNISTON, J T., STREHLER, E. E., FISCHER, R., HEIM, R., VOGEL, G., MATHEWS, S., STREHLER-PAGE, M.-A., JAMES, P., VORHERR, T., KREBS, J. AND CARAFOLI, E. (1988). Complete primary structure of a human plasma membrane Ca 2+ pump J biol. Chem. 263, 14152-14159. VOLPE, P., KRAUSE, K.-H., HASHIMOTO, S., ZORZATO, F , POZZAN, T., MELDOLESEI, J. AND LEW, D. P. (1988) "Calciosome", a cytoplasmic organelle: the inositol 1,4,5-triphosphate-sensitive Ca 2+ store of nonmuscle cells9 Proc. natn. Acad. Sci. U S.A. 85, 1091-1095. WEBER, J. L. (1987). Analysis of sequences from the extremely A+T-rich genome of Plasmodium falciparum Gene 52, 103-109 WEBER, J. L. (1988). Molecular biology of malaria parasites. Expl Parasit. 66, 143-170. YANISH-PERRON, C, VIEIRA, J. AND MESSING, J. (1985). Improved M13 phage cloning vectors and host strains' nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119. (Received 14 May 1990 - Accepted 23 July 1990) Cation-transporting ATPase gene 495