Download Structure of a Plasmodium yoelii gene

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).
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(Received 14 May 1990 - Accepted 23 July 1990)
Cation-transporting ATPase gene
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