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Molecular Biology of the Cell
Vol. 11, 1385–1400, April 2000
A Dibasic Motif in the Tail of a Class XIV
Apicomplexan Myosin Is an Essential Determinant of
Plasma Membrane Localization
Christine Hettmann,* Angelika Herm,* Ariane Geiter,* Bernd Frank,*
Eva Schwarz,
†
Thierry Soldati,
†
and Dominique Soldati*
‡
*Zentrum fu¨r Molekulare Biologie, Universita¨t Heidelberg, D-69120 Heidelberg, Germany;
and
†
Department of Molecular Cell Research, Max-Planck-Institut for Medical Research, D-69120
Heidelberg, Germany
Submitted November 30, 1999; Revised January 19, 2000; Accepted January 19, 2000
Monitoring Editor: Paul T. Matsudaira
Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form
of substrate-dependent locomotion essential for host cell invasion and shown to involve the
parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express
three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and
completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent
structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner.
Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized
right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane
association. Recombinant TgM-D also had a peripheral although not as sharply defined localiza-
tion. Truncation of their respective tail domains abolished peripheral localization and tight
membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was
sufficient to confer plasma membrane localization and sedimentability. The peripheral localization
of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP
chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the
specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed
mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous
plasma membrane receptor and the implications for the invasion process.
INTRODUCTION
The phylum of Apicomplexa comprises numerous patho-
gens of medical and veterinary significance. Among them,
Plasmodium falciparum is the most virulent of the Plasmodium
species responsible for malaria in human. The opportunistic
pathogen Toxoplasma gondii causes diseases in immunocom-
promised patients and in congenitally infected infants. Al-
though members of the Apicomplexa differ significantly in
their host range and cell type specificity, they do share a
similar mechanism for host cell entry. Host cell penetration
is a prerequisite for the survival of these obligate intracellu-
lar parasites and does not occur by induced phagocytosis
but via an active process from the parasites (Sibley, 1995). In
the absence of locomotive organelles, apicomplexan para-
sites have developed an unusual mode of substrate-depen-
dent gliding locomotion that is necessary for invasion (Sib-
ley et al., 1998). The inhibitory functions of cytochalasins on
invasion by Plasmodium (Miller et al., 1979) and Eimeria (Rus-
sell, 1983) have been reported and argue for a participation
of actin filaments. More recently, a direct involvement of T.
gondii actin has been demonstrated, establishing that motil-
ity is essential for invasion and depends on the parasite’s
own cytoskeleton (Dobrowolski and Sibley, 1996). An actin-
based motor is predicted to generate the force during inva-
sion by Apicomplexa, and this hypothesis is corroborated by
the inability of parasites to glide and to invade in the pres-
ence of the inhibitor of myosin heavy chain ATPase butane-
dione monoxime (Dobrowolski et al., 1997a; Pinder et al.,
1998). Similarly, tachyzoites treated with jasplakinolide, a
membrane-permeable actin-polymerizing and filament-sta-
bilizing drug, reversibly inhibit host cell invasion (Shaw and
Tilney, 1999). Members of the myosin superfamily are
mechanoenzymes that convert chemical energy stored in
ATP into a directed force along actin filaments (Spudich,
1994). In the past few years evidence has emerged for roles
played by actin-based molecular motors in a wide range of
‡
Corresponding author. E-mail address: soldati@sun0.urz.uni-
heidelberg.de.
© 2000 by The American Society for Cell Biology 1385
membrane movements (Hasson and Mooseker, 1995; Mer-
mall et al., 1998). In the case of host cell invasion by apicom-
plexan parasites, a capping model proposes that gliding
motility shall be driven by the redistribution of transmem-
brane proteins from the apical to the posterior pole of the
parasite along the subcortical actomyosin system. Corre-
spondingly, the molecular motor powering gliding locomo-
tion and capping is expected to localize right beneath the
plasma membrane. Recently, three myosins were identified
in T. gondii (Heintzelman and Schwartzman, 1997) and
shown to build a 14th phylogenetic and structural class of
myosins (Mermall et al., 1998). Antibodies cross-reacting
with heterologous myosins were used to immunolocalize a
myosin at the anterior pole of T. gondii tachyzoites
(Schwartzman and Pfefferkorn, 1983), and more recent work
from the same group potentially identified this myosin as
TgM-A (Heintzelman and Schwartzman, 1999). In addition,
antibodies raised against the conserved peptide LEAF re-
vealed one or more myosins localized beneath the plasma
membrane and also scattered throughout the cytosol of T.
gondii tachyzoites (Dobrowolski et al., 1997a). Similarly, an-
tibodies generated against a small fragment of the motor
domain of Pf-myo1 showed labeling concentrated under the
plasma membrane of P. falciparum merozoite but absent
from the apical prominence itself (Webb et al., 1996). The P.
falciparum myosin recognized by these antibodies migrates
slightly above 100 kDa in SDS-PAGE, is expressed in mature
schizonts and merozoites, and localizes predominantly
around the periphery of the cell (Pinder et al., 1998).
Here, we report of the identification of a 91-kDa T. gondii
myosin of class XIV, TgM-D, and of the completion of the
PfM-A sequence, the likely P. falciparum homologue of
TgM-A. The exquisite amenability of T. gondii to molecular
genetics allowed us to investigate the determinants of sub-
cellular localization of two class XIV myosins. A pair of basic
residues is essential to target TgM-A to the periphery, de-
fining its cargo-binding site down to the amino acid level.
Such precise mapping has only been achieved for two other
myosins, NINAC, a Drosophila myosin III interacting with
INAD (Wes et al., 1999), and Myo2, a Saccharomyces cerevisiae
myosin V binding to the vacuole (Catlett and Weisman,
1998). In addition, the first purification of such a myosin is
presented, allowing us to show that, despite its structural
divergences, TgM-A had the ability to bind F-actin in an
ATP-dependent manner. Evidence is presented that the pe-
ripheral localization is largely independent of the actin cy-
toskeleton and is likely due to specific and saturable inter-
action with a proteinaceous component of the plasma
membrane.
MATERIALS AND METHODS
Strains and Reagents
The bacterial strains for recombinant DNA techniques were Esche-
richia coli XL1-Blue and XLOR. The helper phage ExAssist, from
Stratagene (La Jolla, CA), was used for the in vivo excision of the
phagemid vectors from the
ZAPII clones. Restriction enzymes
were purchased from New England Biolabs (Beverly, MA). The
secondary antibodies for Western blotting were from Bio-Rad (Her-
cules, CA), and those for immunofluorescence were from BioTrend
(Cologne, Germany).
Growth of Parasites and Isolation of DNA
and RNA
T. gondii tachyzoites (RH strain wild-type and RHhxgprt⫺) were
grown in human foreskin fibroblasts (HFFs) or in vero cells (African
green monkey kidney cells) maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% FCS, 2 mM glutamine, and
25
g/ml gentamicin. Parasites were harvested after complete lysis
of the host cells and purified by passage through 3.0-
m filters and
centrifugation in PBS. Genomic DNA was isolated from purified
parasites by SDS-proteinase K lysis followed by phenol-chloroform,
chloroform extractions and ethanol precipitation (Sibley and Boo-
throyd, 1992). Total RNAs were prepared using RNA Clean from
AGS (Heidelberg, Germany) according to the manufacturer’s in-
structions. P falciparum total RNA was purified from the isolate
FCBR, Columbia grown in vitro (Knapp et al., 1989) and kindly
provided by Dr. H. del Portillo (University of Sao Paulo, Sao Paulo,
Brazil).
PCR Screening
PCR was performed on T. gondii genomic DNA and yielded two
distinct myosin fragments. PCR reactions were carried out in a
GeneAmp PCR System 2400 instrument (Perkin-Elmer, Norwalk,
CT). Primers myo1 and myo5 (corresponding to the conserved
motifs GESGAGKT and LEAFGNAKT, respectively) and the pa-
rameters used for the PCR reaction are described elsewhere
(Schwarz et al., 1999). The PCR products were purified over a spin
column (QIAquick PCR purification kit; Qiagen, Hilden, Germany)
and the whole sample was cloned into PCR Script (Stratagene) as
described by the manufacturer. Double-stranded DNA sequencing
of the products was performed by the dideoxy-termination method
using Sequenase version 2.0 (United Stated Biochemical, Cleveland,
OH) with T3 and T7 primers.
Toxoplasma cDNA and Genomic Library Screening
The digoxigenin system (Boehringer Mannheim, Mannheim, Ger-
many) for nonradioactive labeling and detection of nucleic acids
was used to screen a T. gondii cDNA library. The 300-bp myosin
fragment was digoxigenin-dUTP labeled by PCR and used as a
probe to screen the RH (Elmer Pfefferkorn) cDNA library in
ZAPII
from National Institutes of Health AIDS reagents (kindly provided
by D.S. Roos, University of Pennsylvania, Philadelphia, PA). The
detection of positive clones was achieved by chemiluminescence
with CSPD (Boehringer Mannheim) according to the manufacturer.
Plaques from 15 ⫻10
4
phage were screened, and positive clones
were identified on both duplicates. After two additional cycles of
hybridization, positive clones were excised in vivo, and the insert
sizes were determined by restriction digests. One clone contained a
full-length cDNA corresponding to the previously described TgM-A
and predicting a protein of 93 kDa (Heintzelman and Schwartzman,
1997). The second clone encoded another myosin lacking a start
codon (TgM-D). Further screening of cDNA libraries failed to iden-
tify full-length TgM-D cDNA clones. The missing 5⬘sequences of
TgM-D were finally obtained by sequencing genomic cosmid clones
encompassing the TgM-D locus. The cosmid library used the Su-
perCos vector modified with an SAG1/ble Toxoplasma selection
cassette inserted into its HindIII site. The library was prepared from
aSau3AI partial digestion of RH genomic DNA ligated into the
BamHI cloning site and was kindly provided by D. Sibley and D.
Howe (Washington University, St. Louis, MO). The gene coding for
TgM-D was interrupted by 11 introns of various sizes (our unpub-
lished results). Southern blotting analysis of T. gondii genomic DNA
digested with several restriction enzymes yielded patterns of bands
matching those predicted by the restriction map of the cosmid clone,
indicating that TgM-D is a single-copy gene.
We also obtained and sequenced a cosmid clone encompassing
the TgM-B/C locus (GenBank accession number AF202585). This
confirmed that the two proteins, which have a common N terminus
C. Hettmann et al.
Molecular Biology of the Cell1386
and differ only in their C terminus, are really translated from two
transcripts with alternatively spliced 3⬘ends (as suggested by
Heintzelman and Schwartzman, 1997). This also revealed that the
reported common 5⬘termini were cDNA library cloning artifacts,
resulting from an in frame fusion at an EcoRI site with the 3⬘end of
GRA7. The correct cDNA could then be amplified by reverse tran-
scription (RT)-PCR, cloned, and sequenced.
Identification of a Full-Length P. falciparum Myosin
The sequences of the short and conserved, positively charged tail
domains of TgM-A and TgM-D showed a significant homology with
an expressed sequence tag derived from the closely related organ-
ism P. falciparum (BLAST software search). An RT-PCR strategy
using an antisense primer corresponding to the tail and a sense
primer corresponding to a conserved motif in the head domain
allowed us to amplify, clone, and sequence a large fragment of the
myosin. The sequence corresponded to the partial sequence of a P.
falciparum myosin present in the database (Pfmyo-1). The progress
on the genome sequencing project allowed us to complete and to
confirm the sequence of this myosin (PfM-A, accession number
AF105118). The PfM-A gene contains only two introns clustered
immediately downstream of the AUG, suggesting a possible regu-
latory function of the splicing. The deduced amino acid sequence of
PfM-A predicts a protein of 90 kDa similar to TgM-A and TgM-D
(Figure 1). The full cDNA coding for PfM-A was amplified by
RT-PCR using the Titan RT-PCR system (avian myeloblastosis virus
and Expand High Fidelity; Boehringer Mannheim) according to the
manufacturer’s instructions. The primers used in the PCR reaction
were 5-⬘ccatccatgcatgctgttacaaatgaagaaataaaaacggc-3⬘and 5⬘-ggatc-
cttgagctaccattttttttcttatatgagc-3⬘, and the product of amplification
was cloned into pBluescript (Stratagene).
Construction of T. gondii Expression Vectors
TgM-A and TgM-D were amplified by PCR to introduce a SacI site
after the start codon and a BamHI site before the stop codon, using
the sense oligonucleotide 5⬘-gagctcgcgagcaaggaccacgtct-3⬘com-
bined with reverse oligonucleotide primer 5⬘-ggatccgaacgccggctgaa-
cagtcg-3⬘for TgM-A and 5⬘-gagctcggcacgagcctcttcagt-3⬘combined
with 5⬘-ggatccgcaaaccttgatgcccgctt-3⬘, for TgM-D. The PCR prod-
ucts were cloned into bacterial expression vectors (pET䡠TgM-A and
pET䡠TgM-D) and analyzed for expression of the recombinant pro-
tein in BL21 (DE31, expressing T7 polymerase) after isopropyl-1-
thio-

-d-galactopyranoside induction. Polypeptides of ⬃93 kDa for
TgM-A and 91 kDa for TgM-D were produced (our unpublished
results). Vectors used to express TgM-A and TgM-D in T. gondii
were derived from the pS214SCAT vector containing 5⬘and 3⬘
flanking sequences of the SAG1 gene and pT5RCAT containing
5⬘flanking sequence of the TUB1 gene described previously (Soldati
and Boothroyd, 1995). pS䡠TgM-A was generated by subcloning
TgM-A from pET䡠TgM-A into pS/4R between NsiI and PacI sites
(Soldati and Boothroyd, 1995). The vector pT䡠TgM-D was obtained
by cloning TgM-D coding sequence into NsiI and PacI sites of
pT/5R230 (Soldati and Boothroyd, 1995). In these constructs, both
myosins are fused at the N terminus to a c-myc epitope tag and
seven His residues (these sequences were imported from a modified
pET vector [MQEQKLISEEDLAMAMHHHHHHH]), giving rise to
the mycTgM-A and mycTgM-D proteins. The construct pS䡠TgM-
A⌬tail is identical to pS䡠TgM-A except that sequences correspond-
ing to the last 53 amino acids of TgM-A are lacking. This deletion
was obtained by PCR amplification using the sense primer de-
scribed above and the complementary oligonucleotide 5⬘-ttaattaa-
gaggatccgcattctctctgaatctgc-3⬘. To express full-length TgM-D, a frag-
ment of the genomic clone was amplified by PCR using
oligonucleotides 5⬘-atgcatgagctcgcggcaaaaccggag-3⬘and 5⬘-
cagccaaaaccgaagttcc.3⬘and cloned into the NsiI sites of pTc䡠TgM-D
to generate pT䡠TgM-D. In construct pT䡠TgM-D⌬tail, the last 54
amino acids of TgM-D have been deleted. The EcoRV–PacI fragment
of 1091 bp corresponding to the C terminus of the protein has been
replaced by the fragment corresponding to the C terminus lacking
the tail, obtained by PCR amplification using 5⬘-ttaattaaagccgcac-
caacatctttgct-3⬘as a complementary oligonucleotide.
The plasmid pT䡠GFP was constructed by replacement of chloram-
phenicol acetyltransferase (CAT) by the green fluorescent protein
(GFP) coding sequence between the NsiI and PacI sites in the vector
pT5RCAT. An NsiI site was introduced at the N terminus of the GFP
coding sequence using the oligonucleotides 5⬘-ggcgatgcatagtaaag-
gagaagaacttttc-3⬘, and PstI and PacI sites were introduced at the C
terminus using the complementary oligonucleotides 5⬘-gggcttaatta-
agcaccgcctgcagctttgtatagttca-3⬘. The GFP mutant used in this work
is fluorescent as nonfusion protein in tachyzoites (M. Soete, C.
Hetlmann, and D. Soldati, unpublished data) and has been exten-
sively modified in the codon usage (Haseloff et al., 1997). The
constructs pT䡠GFP䡠TgM-Atail, pT䡠GFP䡠TgM-Dtail, and pT䡠GFP䡠PfM-
Atail were generated by cloning the PCR products corresponding to
the last 82 amino acids of TgM-A (as in Figures 7 and 9, or 62, as in
Figures 1A, 5, and 8), the last 57 amino acids of TgM-D, and the last
85 amino acids of PfM-A, respectively, using PstI and PacI sites. The
site-specific mutagenesis of TgM-Atail was achieved by PCR amplification
using the following primers: 5⬘-gcgtactacgctggcatactccacgcggcgcag-
ctgctgaaaaag-3⬘,5⬘-aagaagcagctgctggcagcgacccccttcatcatt-3⬘,5⬘-gcccagg-
ctcacatcgccgcacacctggtggacaac-3⬘,5⬘-cggttaattaaaccaggtgtctgcggatgtg-3⬘,
and 5⬘-gccttaattaagcgcgaatgatgaaggggg-3⬘for TgM-Atail-mut I, -mut II,
-mut III, -⌬14, and -⌬22, respectively. The PCR products of the mutated
tails were cloned in pT䡠GFPdhfrtsHXGPRT between PstI and PacI sites.
The fragment coding for GFP fused to TgM-Atail was cut from
pT䡠GFP䡠TgM-Atail and introduced into pUHD15-1 (Gossen and Bujard,
1992), using EcoRI and BamHI as cloning sites to generate pUHD15-
1䡠GFP䡠TgM-Atail. The vector for nonfusion GFP expression, pUHD15-
1GFP, was kindly provided by R. Lo¨w (ZMBH, Heidelberg, Germany).
Generation of a Serum Specific for the Tail of
TgM-A
Two peptides covering the tail of TgM-A (CVLEAYYAGRRH-
KKQLLKKTP and AHIRRHLVDNNVSPATVQPAFC) were synthe-
sized and coupled to keyhole limpet hemocyanin according to the
instructions of the manufacturer (Pierce, Rockford, IL). Two rabbits
were first immunized with 400
g of both peptides with the com-
plete Freund’s adjuvant. Four successive subcutaneous injections
with 200
g of both peptides in incomplete adjuvant LQ (Gerbu,
Heidelberg, Germany) were performed at 24-d intervals. The serum
was immunoaffinity purified against the peptides previously cou-
pled to Affi-Gel 10 according to the manufacturer (Bio-Rad). Four
milligrams of each peptide were used to affinity purify 2 ml of sera.
Parasite and HeLa Cell Transfection and Selection
of Transformants
T. gondii tachyzoites (RHhxgprt
⫺
) were transfected by electropora-
tion as previously described. Selection of stable transformants ex-
pressing pS䡠TgM-A and pT䡠GFP䡠TgM-Atail were achieved by co-
transfection of the expression vector with the selectable plasmid
(pminHXGPRT). Restriction enzyme-mediated integration was ap-
plied as previously described (Black et al., 1995). The hypoxanthine-
xanthine-guanine-phosphoribosyltransferase (HXGPRT) was used
as a positive selectable marker gene in the presence of mycophenolic
acid and xanthine as described (Donald et al., 1996). Freshly released
parasites (5 ⫻10
7
) of the RHhxgprt
⫺
strain were resuspended in
cytomix buffer in the presence of 100 U of BamHI and were cotrans-
fected with 10–20
g of the plasmid pminHXGPRT, which carries
the HXGPRT gene, and 80–100
g of the plasmids expressing GFP
fusions or myosins. Parasites expressing pT䡠TgM-D, pT䡠GFP䡠TgM-
Dtail, and pT䡠GFP䡠PfM-Atail could not be obtained by cotransfec-
tion; therefore, the selectable marker gene dhfrtsXHGPRTdhfrts was
introduced into the expression vector in a unique SacII site before
selection. HeLa cells were transfected with pUHD15-1䡠GFP䡠TgM-
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1387
C. Hettmann et al.
Molecular Biology of the Cell1388
Atail and by a calcium phosphate method as described previously
(Graham and Eb, 1973).
Cytochalasin D Treatment
Cytochalasin D (Sigma, St. Louis, MO) was stored as DMSO stock at
⫺20°C. Intracellular parasites grown for 24 h in HFFs on glass
coverslips were incubated with 10
g/ml cytochalasin D in medium
at 37°C for up to 3 h. At the indicated time, the coverslips were
taken out and rinsed twice with PBS, and cells were fixed as de-
scribed below.
Indirect Immunofluorescence Microscopy and
Detection of GFP in T. gondii
All manipulations were carried out at room temperature. Intra-
cellular parasites grown for 24 h in HFFs on glass slides were
fixed with 4% paraformaldehyde and 0.05% glutaraldehyde for
20 min. After fixation, slides were rinsed in PBS and 0.1 M
glycine. Cells were then permeabilized in PBS and 0.2% Triton
X-100 for 20 min and blocked in the same buffer with 2% FCS.
Slides were incubated for 60 min with primary antibodies diluted
in PBS and 1% FCS, washed, and incubated for 60 min with
Alexa488- or FITC-labeled goat anti-mouse immunoglobulin Gs
diluted in PBS and 1% FCS. After cytochalasin D treatment,
stainings were carried out with the following modifications. Fix-
ation was with 4% paraformaldehyde only, all subsequent buffers
contained 5 mM EGTA to chelate Ca
2⫹
and avoid disruption of
actin filaments by gelsolin-like activities, and 2% BSA was used
as blocking reagent. Staining of F-actin was performed with
Oregon Green-phalloidin (Molecular Probes, Eugene, OR) for 60
min. The rapid freezing combined with fixation and permeabili-
zation in ultracold methanol was performed as described (Neu-
haus et al., 1998). Slides were mounted in Vectashield (Vector
Laboratories, Burlingame, CA) and kept at 4°C in the dark. The
mAb anti-myc was an ascites preparation of 9E10 used at the
dilution 1:1000. The mAb DG52, recognizing SAG1, coupled to
biotin was generously provided by Dr. J.F. Dubremetz (Institut
Pasteur de Lille). Intracellular parasites expressing GFP were
fixed according to the above protocol and mounted immediately
for microscopic analysis. Confocal images were collected with a
Leica (Nussloch, Germany) laser scanning confocal microscope
(TCS-NT DM/IRB) using a 100⫻Plan-Apo objective, numerical
aperture 1.30. Single optical sections were recorded with an
optimal pinhole of 1.0 (according to Leica’s instructions) and 16
times averaging. All other micrographs were obtained with a
Zeiss (Thornwood, NY) Axiophot microscope with a camera
(CH-250; Photometrics, Tucson, AZ). Adobe Photoshop (Adobe
Systems, Mountain View, CA) was used for image processing.
Western Analysis of Parasite Lysates
SDS-PAGE was performed using standard methods (Leammli,
1970). Crude extracts from T. gondii tachyzoites were separated by
SDS-PAGE and transferred to nitrocellulose. Western blot analysis
was carried out essentially as described previously (Soldati et al.,
1998), using 8–12% polyacrylamide gels run under reducing condi-
tion with 144 mM

-mercaptoethanol or 100 mM DTT in the loading
samples. After electrophoresis, proteins were transferred to Hybond
ECL nitrocellulose (Amersham, Arlington Heights, IL). For detec-
tion, the membranes were incubated with the mAb 9E10 (mouse
ascites fluid diluted 1:1000 in PBS and 0.5% Tween 20) and then
with the affinity-purified horseradish peroxidase-conjugated goat
anti-mouse immunoglobulin G (1:2000), and bound antibodies were
visualized using the ECL system (Amersham). Direct recording of
chemoluminescent signals and densitometry by an LAS-1000 lumi-
nescent image analyser (Fuji, Tokyo, Japan) allowed quantification
of signal intensities within a broad linear range.
Cell Fractionation
Freshly released parasites (10
8
) from infected cells were resus-
pended into 300
l of buffer and lysed by freezing and thawing
three times followed by sonification (four times for 30 s on ice).
Pellet and soluble fractions were separated by ultracentrifugation
1 h at 55,000 rpm at 4°C. The buffers were PBS, PBS and 1 M NaCl,
PBS and 0.1 M Na
2
CO
3
, pH 11.5, PBS and 2% Triton X-100, and 3 M
urea. In addition, all buffers contained 5 mM ATP to minimize the
interaction of myosins with F-actin, and 5 mM EDTA.
Purification of Recombinant Myosin from T. gondii
Freshly lysed transgenic parasites expressing TgM-A⌬tail (10
10
par-
asites) were lysed under native conditions by freezing and thawing
followed by sonification. The soluble fraction was separated by
ultracentrifugation at 65,000 rpm, and the supernatant was applied
to an Ni-nitrilotriacetic acid resin column and eluted according to
the manufacturer (The QIAexpressionist; Janknecht et al., 1991);
determination of protein concentration was performed by a Brad-
ford assay (Bio-Rad).
Actin Binding Assays
The binding of TgM-A⌬tail to F-actin was measured using an F-
actin sedimentation assay as described previously (Jung and Ham-
mer, 1994). F-actin from rabbit skeletal muscle was purified accord-
ing to the method of Pardee and Spudich (1982). Binding assays
were performed using a 4
M final concentration of F-actin and 0.5
g of TgM-A⌬tail (corresponding to ⬃0.06
M), The buffer (plus
ATP) contained 10 mM Tris-HCl, pH 7.5, 130 mM KCl, 2 mM MgCl
2
,
2 mM ATP, and 0.1 mM DTT. The washing steps were performed by
resuspending the pellet in washing buffer and were followed by
centrifugation at 50,000 rpm. Pellets and supernatants were ana-
lyzed by SDS-PAGE and Coomassie blue staining.
RESULTS
Identification and Cloning of Two Members of the
Class XIV Myosins
To identify myosins in T. gondii, we designed a PCR screen-
ing strategy taking advantage of the highly conserved motifs
present in the head domain (Schwarz et al., 1999). The ap-
proach identified TgM-A, a myosin already described by
Heintzelman and Schwartzman (1997), and TgM-D, an ad-
ditional member of the apicomplexan-specific class XIV of
Figure 1 (facing page). TgM-D is a member of the class XIV
myosins. (A) Sequence alignment of the class XIV myosins. Identical
amino acids are indicated by a star; homologies are indicated by a
dot. The TgM-A glutamine 419 corresponds to the TEDS site, and
the serine 693 is found instead of the conserved glycine proposed to
act as a pivot point of the lever arm. (B) Phylogenetic tree with 51
myosins representative of each known class. TgM-D and the com-
pleted PfM-A belong to the class XIV. (C) Schematic presentation of
the constructs used in this study. The numbers on top of the bars
indicate the corresponding residues from each myosin (see A). Most
accession numbers can be found at http://www.mrc-lmb.cam-
.ac.uk/myosin/trees/accession.html, and some can be found in an
article by Schwarz et al. (1999). Accession numbers for the apicom-
plexan myosins: TgM-A, AF006626; TgM-B, AF006627; TgM-C,
AF006628; a genomic fragment with the correct 5⬘end of TgM-B and
-C cDNAs, AF202585; TgM-D, AF105118; TgM-E, AF221131; PfM-A,
AF105117; PfM-B, AF222716; and PfM-C, AF222717.
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1389
myosins (Figure 1B). The entire TgM-D gene was sequenced.
The protein predicted from the open reading frame has 823
amino acid residues (Figure 1A) with a mass of 91 kDa
(accession number AF105117).
The sequences of the short and conserved, positively
charged tail domains of TgM-A and TgM-D showed a sig-
nificant homology with an expressed sequence tag derived
from the closely related organism P. falciparum. This allowed
us to identify and subsequently clone a complete myosin
corresponding to a partial P. falciparum sequence present in
the database (PfMyo-1; see MATERIALS AND METHODS).
We named this myosin PfM-A (Figure 1A) because its pre-
dicted sequence of 90 kDa has highest homology to TgM-A
(Figure 1B). The degree of conservation at the amino acid
level between the myosins is high and extends throughout
the entire coding region. There is 55.7% identity between
TgM-A and TgM-D, 63.1% between TgM-A and PfM-A, and
52.0% between TgM-D and PfM-A. Pairwise comparisons
between any of these myosins with the other two, alterna-
tively spliced, class XIV myosins from T. gondii, TgM-B and
-C, give lower identity scores of ⬃47–49%.
Like the other apicomplexan myosins TgM-D and PfM-A
have a very short tail, do not contain the strictly conserved
glycine residue at the proposed fulcrum point of the lever
arm, and appear to lack conserved “IQ” motifs that bind
calmodulin and calmodulin-related proteins. Among their
particularities, and contrary to the P. falciparum molecule, all
the T. gondii myosins do not follow the TEDS rule (Heint-
zelman and Schwartzman, 1997; Figure 1A), which describes
the presence of an acidic or phosphorylatable residue at a
precise position close to the actin-binding region (originally
mapped by Brzeska et al., 1989, 1990; for review, see Bement
and Mooseker, 1995). In lower eukaryotes, phosphorylation
of this conserved serine or threonine was shown to be crucial
for the stimulation of the ATPase activity of class I myosins
(Bement and Mooseker, 1995; Carragher et al., 1998; Novak
and Titus, 1998). It is unclear at the moment whether and
how these class XIV motors are activated and how confor-
mational changes in the molecules occur.
The different recombinant constructs of TgM-A and
TgM-D used in this study are presented schematically in
Figure 1C.
Antibodies to the Tail of TgM-A Are Isoform
Specific
To investigate the expression and distribution of TgM-A, we
raised polyclonal sera against two peptides covering its
entire tail. The specificity of the antibodies was assessed by
Western blotting of parasites expressing GFP䡠TgM-Atail and
GFP䡠TgM-Dtail (Figure 2A). The anti-myc antibodies de-
tected both GFP chimeras, but despite high overall sequence
homologies, the anti-tail antibodies recognized only the
TgM-A tail (Figure 2A). In wild-type parasites, the affinity-
purified antibody recognized a single band migrating above
90 kDa, the predicted size of TgM-A (Figure 2B). Recombi-
nant mycTgM-A was detected by the anti-tail antibodies and
was slightly bigger than endogenous TgM-A. Confirming
the antibody specificity, only the endogenous protein was
detectable in parasites expressing mycTgM-A⌬tail (Figure
2B).
The Expression of Endogenous TgM-A Is Down-
regulated in the Transgenic Parasites Expressing
Full mycTgM-A
Surprisingly, in recombinant parasites expressing full-length
mycTgM-A, the endogenous TgM-A was barely detectable
(Figure 2B). To obtain a quantitative impression of the phe-
nomenon, equal numbers of wild-type and different recom-
binant parasites were analyzed (Figure 2B). MycTgM-A ap-
peared to be expressed at approximately two- to threefold
the level of endogenous protein found in wild-type parasites
and parasites expressing mycTgM-A⌬tail. We conclude that
expression of full-length mycTgM-A, but not of the head
domain alone, led to down-regulation of the endogenous
TgM-A protein and expression. In parasites expressing
GFP䡠TgM-Atail, the anti TgM-A tail antibodies simulta-
neously detected the 40-kDa GFP chimera and the endoge-
nous TgM-A. Expression of the endogenous protein ap-
peared slightly reduced compared with wild-type cells,
indicating that the tail alone mimicked the down-regulation
effect observed with the full-length myosin. Because the
signal for the GFP chimera was approximately half as in-
tense as for mycTgM-A (Figure 2B, first lane), we suggest
that the effect is partial probably because of a lower expres-
sion level.
The Presence of Its Tail Influences the Expression of
TgM-D
TgM-D was well expressed transiently, but our initial efforts
to obtain stable transformants were unsuccessful, potentially
because mycTgM-D overexpression was not well tolerated
by the parasites. To circumvent this problem, we introduced
the selectable marker directly in the expression vector and
obtained a few stable cell lines expressing mycTgM-D. Ex-
Figure 2. Immunoblot analysis of wild-type and recombinant par-
asites. (A and B) The antibody against the tail of TgM-A does not
recognize the tail of TgM-D. Extracts of parasites expressing
GFP䡠TgM-Atail or GFP䡠TgM-Dtail were blotted with anti-myc anti-
bodies (mAb 9E10) and with anti-TgM-A tail antibodies (A). (B) The
expression of mycTgM-A down-regulates the level of endogenous
protein. Detection of endogenous and recombinant TgM-A with an
antibody directed against its tail is shown. The lanes were loaded
with similar amounts of freshly lysed wild-type parasites or para-
sites expressing mycTgM-A, mycTgM-A⌬tail, or GFP䡠TgM-Atail.
C. Hettmann et al.
Molecular Biology of the Cell1390
pression of transgenic mycTgM-D (Figure 3B) was signifi-
cantly reduced compared with mycTgM-A (Figure 3A), al-
though the promoter used to drive its expression was
previously shown to be stronger (Soldati and Boothroyd,
1995). The construct pT䡠TgM-D⌬tail could readily be intro-
duced into the parasites by simple cotransfection with the
selection marker plasmid. Immunoblot analysis of total cell
lysates with an antibody against the c-myc epitope showed
that parasites transformed with pS䡠TgM-A or pT䡠TgM-D
expressed a single protein of the expected sizes of 93 and 91
kDa (Figure 3). Deletion of their tails resulted in slightly
higher mobility. A semiquantitative analysis was performed
by comparing the signal intensities resulting from loading
lanes with a precise number of parasites. This revealed that
mycTgM-A and mycTgM-A⌬tail were expressed at compa-
rable levels (Figure 3A), whereas mycTgM-D⌬tail was ex-
pressed at an ⬃5- to 10-fold higher level than mycTgM-D
(Figure 3B). These results provided evidence that the pres-
ence of the tail ofTgM-D limits the expression level of the
recombinant protein.
TgM-A Localization Is Confined to the Parasite
Periphery
TgM-A and TgM-D share a high degree of homology, and it
is not yet known whether T. gondii expresses additional
similar myosins (in addition to the related TgM-B and -C,
which harbor longer and different tail domains). To deter-
mine unambiguously, without risk of cross-reaction, the
subcellular localization of TgM-A, TgM-D and mutants
thereof, two approaches were used. First we generated iso-
form-specific antibodies. Second, we used an epitope-tag-
ging strategy, taking advantage of the easy accessibility of T.
gondii to genetic manipulation. The anti-myc tag antibody
revealed that, aside from a relatively diffuse cytoplasmic
distribution, a proportion of mycTgM-A localized precisely
beneath the plasma membrane (Figure 4A, a), as visualized
by the almost perfect colocalization with SAG1, the major
surface antigen of T. gondii tachyzoites (Figure 4A, b). Be-
cause SAG1 is anchored at the plasma membrane via a
glycosylphosphatidylinositol, the staining suggests that
TgM-A associates closely with this membrane, although the
resolution of light microscopy cannot exclude that the my-
osin interacts with the inner membrane complex. This latter
structure is composed of flattened membrane cisternae
found in close apposition with the plasma membrane of all
Apicomplexa. It covers the elaborate basket of microtubules
and contributes to the maintenance of cell shape and polar-
ity (Morrissette et al., 1997).
Surprisingly, despite strong and specific signals obtained
on Western blots (Figure 2), imunofluorescence using the
anti-TgM-A tail antibodies failed to detect endogenous
TgM-A in wild-type parasites (our unpublished results) and
parasites expressing mycTgM-A⌬tail (Figure 4B, e) and also
failed to corroborate the peripheral localization of my-
cTgM-A (Figure 4B, compare a and b). Nevertheless, in the
latter case a signal was clearly perceived and was similar in
intensity (but not in staining pattern) to the cytoplasmic
signal obtained for mycTgM-A⌬tail (Figure 4B, compare d
and b). It is reasonable to assume that the tail of TgM-A,
being very rich in arginine and lysine residues, aldehyde
cross-linking to plasma membrane proteins, may lead to
complete epitope masking. Therefore, we used an alterna-
tive fixation method involving rapid freezing fixation and
permeabilization in ultra-cold methanol, an ideal method to
preserve both structure and antigenicity (Neuhaus et al.,
1998). Figure 4C illustrates that both the endogenous TgM-A
(Figure 4C, b) and the myc-tagged protein (Figure 4C, a)
were now detected by the anti-tail antibody. As expected,
the signal for TgM-A was slightly lower in wild-type para-
sites than in recombinants expressing the myc-tagged pro-
tein. The cytoplasmic staining for mycTgM-A (Figure 4B, b)
was stronger than for endogenous TgM-A (Figure 4B, e).
Finally, irrespective of the expression level, both proteins
localized similarly all around the parasite periphery, con-
firming the epitope-tagging data.
The Short Tail Domains Are Necessary for Plasma
Membrane Localization of the T. gondii Myosins
Myosin molecules are modular motors made up of three
domains. The N-terminal domain is the actin binding motor
unit per se. The middle neck domain bears the light chains
and acts as a lever arm. The tail domain is exceptionally
divergent and reflects the diversity in myosin functions. The
tail is thought to target a given myosin to its cargo or site of
action and thereby to determine the specific task of the
motor. To assess the role of the short tail domains in the
subcellular distribution of the two proteins, we constructed
vectors expressing myosins lacking their tail. Recombinant
parasites expressing mycTgM-A⌬tail and mycTgM-D⌬tail
were analyzed by confocal microscopy (Figure 4, B and D).
Figure 3. The presence of its tail limits the expression level of
recombinant TgM-D. Western blot analysis used the anti-myc anti-
body on parasites expressing recombinant mycTgM-A and my-
cTgM-A⌬tail (A) or mycTgM-D and mycTgM-D⌬tail (B). The re-
spective expression levels of each full-length and truncated myosin
were judged by comparing the signals resulting from the loading of
105, 106, or 107 parasites, respectively. MycTgM-A and mycTgM-
A⌬tail,as well as mycTgM-D⌬tail, were expressed at comparable
levels, whereas full-length mycTgM-D was expressed at an approx-
imately 10-fold reduced level.
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1391
Figure 4. Immunofluorescence localization of TgM-A and TgM-D and their respective tail-less constructs. Classical (A and D) and confocal (B and
C) immunofluorescence microscopic analysis of wild-type parasites (C, b) or parasites expressing mycTgM-A (A, B, a–c, and C, a), mycTgM-A⌬tail
(B, d–f), mycTgM-D (D, a), or mycTgM-D⌬tail (D, b) is shown. Fixation was performed either by paraformaldehyde-glutaraldehyde (A, B, and D)
or ultracold methanol (C). (A) MycTgM-A, visualized by anti-myc antibodies (a), colocalized at the parasite periphery (c) with SAG1, the major
glycosylphosphatidylinositol-anchored surface antigen of T. gondii (b). (B and C) The antibody against the tail of TgM-A did not detect TgM-A (B,
e) or mycTgM-A (B, b) at the plasma membrane when cells were fixed by paraformaldehyde-glutaraldehyde, even though mycTgM-A was detected
at the periphery by anti-myc staining (B, a). The rapid freezing and fixation in ultracold methanol allowed peripheral localization of endogenous
TgM-A (C, b) and mycTgM-A (C, a). Myc-TgM-A⌬tail was mainly found in the cytoplasm (B, d). The anti-tail antibodies detected a cytosolic pool
of mycTgM-A (B, b). (D) As detected by anti-myc antibodies, mycTgM-D was enriched at the parasite periphery (a) but in a way distinct from
TgM-A (compare with A–C). MycTgM-D⌬tail was mainly cytoplasmic (b).
C. Hettmann et al.
Molecular Biology of the Cell1392
As already observed above, absence of tail in mycTgM-
A⌬tail caused the disappearance of the membrane-associ-
ated pool (Figure 4B, d).
In a way similar to mycTgM-A, mycTgM-D appeared to
distribute predominantly at the parasite periphery (Figure
4D, a), even though not as sharply as mycTgM-A. The subtle
difference in distribution may indicate a different mode or
mechanism of localization. As in the case of mycTgM-A, the
absence of tail caused an apparent redistribution of my-
cTgM-D to the cytoplasm (Figure 4D, b), and, as mentioned
above (Figure 3B), it also led to a significant increase in the
level of the recombinant product compared with the full-
length protein.
The Tail of TgM-A Is Necessary for Distribution in
the Particulate Fraction
To corroborate the localization data with biochemical frac-
tionation, cells were first lysed in PBS, separated into soluble
and sedimentable fractions by high-speed centrifugation,
and analyzed by Western blotting. A comprehensive analy-
sis was undertaken on cells expressing GFP䡠TgM-Atail, al-
lowing for simultaneous investigation of endogenous
TgM-A and of the GFP tail chimera. In agreement with the
imunofluorescence, TgM-A partitioned with the particulate
fraction (Figure 5A). The nature of this interaction was fur-
ther investigated. TgM-A behaved as a strongly associated
peripheral membrane protein, because it was resistant to
solubilization by high salt. Full solubilization was achieved
only by carbonate treatment (pH 11.5) and extraction by
detergent (1% Triton X-100) or 3 M urea (our unpublished
results).
Even though mycTgM-A⌬tail appeared cytoplasmic (Fig-
ure 4B, d), it is formally possible that TgM-A localized at the
periphery and, despite inclusion of ATP during fraction-
ation, interacted with the particulate fraction through bind-
ing of its head domain to the actin cytoskeleton. Therefore,
the role of the tail domain was investigated for recombinant
TgM-A (Figure 5C). When parasites were lysed in PBS,
about one-third to one-half of mycTgM-A sedimented with
the particulate, membrane fraction whereas mycTgM-A⌬tail
partitioned essentially with the soluble cytosolic fraction, in
perfect agreement with the imunofluorescence data. This
indicated that the sedimentation of TgM-A was not due to
interactions through the motor domain but was likely me-
diated by the tail domain (also see Figure 7 and accompa-
nying text). Similar results were obtained for mycTgM-D
(Figure 5C). Moreover, whereas endogenous TgM-A is
quantitatively recovered with the particulate fraction (Fig-
ure 5, A and B), approximately half of mycTgM-A, which is
overexpressed two- to threefold, is found in the soluble
fraction (Figure 5C), possibly because a plasma membrane
receptor is saturated.
TgM-A Binds to F-actin in an ATP-dependent
Manner
The peripheral targeting of TgM-A appears not to depend
on the interaction of the head domain with the actin
cytoskeleton. This could potentially be caused by the fact
that the motor domains of class XIV myosins are pre-
dicted to exhibit important structural divergences. There-
fore, it was crucial to assess experimentally whether these
proteins truly act as ATP-driven, actin-dependent motors.
In addition, according to the capping model of invasion,
the myosin involved would have to be able to grab onto
F-actin and exert ATP-dependent traction. The complete
solubility of recombinant mycTgM-A⌬tail in tachyzoites
allowed us to purify biochemical quantities of the protein
under native conditions. The degree of purity of the re-
combinant mycTgM-A⌬tail was examined by SDS-PAGE
and Coomassie blue staining (Figure 6A). The identity of
two low-molecular-mass bands (⬃30 kDa) has not been
fully determined yet (our unpublished results), but they
were distinct from degradation products of TgM-A and
could potentially represent copurifying light chain(s). To
Figure 5. Distribution of endogenous and recombinant myosins
studied by subcellular fractionation. Cells expressing GFP (B) or
GFP䡠TgM-Atail (A) were lysed in the presence of 5 mM ATP under
different conditions and separated by high-speed centrifugations in
soluble (S) and particulate (P) fractions. Cells were lysed in PBS, in
a low-osmolarity buffer (PBS diluted 1:10), in PBS with 1 M NaCl, in
0.1MNa
2
CO
3
, pH 11.5, or in PBS with 1% Triton X-100. The
distribution of endogenous TgM-A and either GFP䡠TgM-Atail or
GFP was determined simultaneously by immunoblotting with an-
tibodies against the tail of TgM-A either alone (A) or combined with
anti-myc antibodies (B). (C) The distribution of mycTgM-A, my-
cTgM-A⌬tail, and mycTgM-D was detected by anti-myc antibody
after lysis in PBS.
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1393
gain first insights into the biochemical characteristics of
this mycTgM-A⌬tail, a cosedimentation assay with F-ac-
tin was performed (Figure 6B). The results showed that
mycTgM-A⌬tail coprecipitated with actin only in the ab-
sence of ATP and that the sedimented myosin was sub-
sequently, even though only incompletely, released by the
addition of 10 mM ATP, demonstrating that this purified
myosin was able to reversibly bind actin in an ATP-
dependent manner. Similar results have been obtained
with full-length mycTgM-A (our unpublished results).
Together, our results also demonstrated the functionality
of both recombinant TgM-A constructs.
The Short TgM-A and TgM-D Tail Domains Are
Sufficient to Target a Reporter Protein to the Cell
Periphery
The short basic tails of TgM-A, TgM-D and PfM-A are very
similar in length and in amino acid composition. A compar-
ison of the myosin tails is depicted in Figure 7A. The con-
served spots of basic residues are highlighted. Analysis of
the predicted secondary structure of these domains also
revealed a conserved overall organization in three helices
interrupted by short loops. To determine whether the short
tail domains are sufficient to confer specific cellular distri-
Figure 6. MycTgM-A⌬tail sediments
with F-actin in an ATP-dependent
manner. (A) SDS-PAGE and Coomas-
sie blue staining of mycTgM-A⌬tail
purified from an extract of recombi-
nant parasites. (B) SDS-PAGE analysis
and Coomassie blue staining of the su-
pernatant (S) and pellets (P) obtained
after cosedimentation of mycTgM-
A⌬tail with F-actin in the presence (⫹)
or absence (⫺) of ATP. MycTgM-
A⌬tail was completely soluble in the
absence of F-actin (Control). MycTgM-
A⌬tail could be released from F-actin
by subsequent incubation with ATP.
Rabbit muscle F-actin was used at 4
M and ATP at 10 mM in the presence
of 130 mM KCl.
Figure 7. The tails of TgM-A and TgM-D are sufficient to confer to GFP both peripheral localization and association with the particulate
fraction. (A) Alignment of three highly related apicomplexan myosin tails. The basic residues are boxed. (B) Localization of GFP (a),
GFP䡠TgM-Atail (b), GFP䡠TgM-Dtail (c), and GFP䡠PfM-Atail (d). Fluorescence was observed directly (a and b) or after indirect immunofluo-
rescence with anti-myc antibodies (c and d). (C) Recombinant parasites expressing GFP, GFP䡠TgM-Atail, GFP䡠TgM-Dtail, and GFP䡠PfM-Atail
were lysed in PBS and separated by high-speed centrifugations in soluble (S) and particulate (P) fraction. The partitioning of GFP and the
tail chimeras reflected their respective intracellular localization observed by imunofluorescence microscopy.
C. Hettmann et al.
Molecular Biology of the Cell1394
bution to the myosins, the tails of TgM-A (last 82 amino
acids) TgM-D (last 57 amino acids), and PfM-A (last 85
amino acids) were fused to GFP. The plasmids pT䡠GFP and
pT䡠GFP䡠TgM-Atail were stably integrated into tachyzoites
by cotransfection with HXGPRT expression vector. We had
to introduce the selectable marker gene on pT䡠GFP䡠TgM-
Dtail and pT䡠GFP䡠PfM-Atail to obtain stable transformants.
The transgenic parasites expressing GFP and GFP䡠TgM-Atail
were analyzed by direct GFP fluorescence, whereas
GFP䡠TgM-Dtail and GFP䡠PfM-Atail required indirect immu-
nofluorescence analysis using anti-myc antibodies to defi-
nitely visualize the recombinant protein (Figure 7B). The
nonfusion GFP was abundantly expressed, homogeneously
distributed in the cytosol with a significant accumulation in
the nucleus (Figure 7B, a). In contrast, GFP䡠TgM-Atail was
found almost exclusively closely associated with the plasma
membrane, with barely detectable cytosolic staining (Figure
7B, b). In a way reminiscent of the respective localization of
the full-length proteins, GFP䡠TgM-Dtail showed a slightly
more diffuse and speckled peripheral signal than the
GFP䡠TgM-Atail chimera (Figure 7B, c). The difficulty re-
ported above to express high amounts of mycTgM-D ap-
peared indeed to be dependent of the tail domain, because
parasites expressing GFP䡠TgM-Dtail were also difficult to
obtain, and the fusion was expressed at a very reduced level
compared with both GFP and GFP䡠TgM-Atail. This was
visible on the immunofluorescence stainings and was con-
firmed by Western blotting (our unpublished results). Sim-
ilarly, the GFP䡠PfM-Atail fusion was expressed at a low level
and localized essentially to the parasite cytosol (Figure
7B, d).
The GFP䡠TgM-Atail and GFP䡠TgM-Dtail Chimeras
Partition with the Membrane Fraction
The transgenic parasites expressing GFP and chimeras were
lysed in PBS, separated into soluble and particulate frac-
tions, and subsequently analyzed by Western blot. As illus-
trated in Figure 7C, the migration on SDS-PAGE of the GFP
chimera was in agreement with their predicted sizes.
GFP䡠TgM-Atail and GFP䡠TgM-Dtail completely partitioned
with the particulate fraction, whereas GFP and GFP䡠PfM-
Atail were entirely recovered in the supernatant. The quan-
titative association of GFP with the particulate fraction was
comparable with the behavior of endogenous TgM-A. In
contrast, GFP䡠TgM-Dtail sedimented despite the fact that the
main part of mycTgM-D was soluble. This could potentially
indicate that both GFP chimeras were expressed at levels
that did not saturate their respective peripheral binding
sites. In addition, it is important to note that both chimeras
were fully solubilized by carbonate treatment, demonstrat-
ing that they were not simply aggregated in the cell (Figure
7C). In fact, the solubilization characteristics of GFP䡠TgM-
Atail were extremely similar to the ones of endogenous
TgM-A (see Figure 5A).
The Localization of TgM-A Is Not Due to
Interactions with the Actin Cytoskeleton
Altogether, the data indicate a strong interaction of the tail
of TgM-A with components of the parasite cortex, likely
with proteins associated peripherally with the plasma mem-
brane, even though an interaction with the closely apposed
inner membrane complex cannot be excluded. To defini-
tively eliminate the potential targeting role of the cortical
actin cytoskeleton, F-actin was severely compromised by
cytochalasin D incubation. It has been previously reported
that phalloidin fails to stain the actin filaments in T. gondii
(Dobrowolski et al., 1997b). Therefore, as indication of the
treatment efficacy, the peripheral localizations of mycTgM-A
(Figure 8A, a and b) and GFP䡠TgM-Atail (Figure 8A, c and d)
are shown simultaneously with a phalloidin staining of the
host actin cytoskeleton. No noticeable change of localization
of both mycTgM-A and GFP䡠TgM-Atail was observed de-
spite severe disturbance of the actin cytoskeleton (Figure 8A,
compare b with a and d with c).
The Tail of TgM-A Does Not Localize to the Plasma
Membrane of HeLa Cells
Stretches of basic residues in the tail of class I myosins have
been shown to contain high-affinity phospholipid binding
sites (Doberstein and Pollard, 1992). If TgM-A were to inter-
act with the plasma membrane solely through unspecific
interaction with lipids, the peripheral localization should be
observed in unrelated cellular systems. When expressed in
HeLa cells (Figure 8B), GFP䡠TgM-Atail is homogeneously
distributed in the cytoplasm, confirming that the association
with the particulate fraction in T. gondii was not due to
improper folding. More importantly, the chimera showed no
sign of membrane association. Indeed, its cytoplasmic local-
ization (Figure 8B, d) was indistinguishable from the one of
GFP (Figure 8B, b).
Identification of the Molecular Determinants of
Peripheral Membrane Localization
To define precisely, at the amino acid level, the determinants
of membrane localization, deletion analysis and site-specific
mutagenesis of the basic residues in the tail of TgM-A fused
to GFP were undertaken (Figure 9A). Faithful expression
and stability of the respective constructs were confirmed by
immunoblotting (Figure 9C). The GFP fusions with mutants
of TgM-A tail were also examined by indirect immunofluo-
rescence (Figure 9B). Deletion of the C-terminal extension
composed of the last 14 amino acids (TgM-Atail⌬14) did not
alter the association of mycTgM-A with the cell membrane.
In contrast, a further deletion encompassing the last 22
amino acids (TgM-Atail⌬22) caused a complete loss of mem-
brane localization. The mutagenesis of two arginine residues
within the last 22 amino acids into alanine residues (TgM-
Atail mut III) completely abolished localization. However,
the conversion of the three other sets of basic residues into
neutral amino acids (TgM-Atail mut I and TgM-Atail mut II)
did not alter significantly the plasma membrane distribu-
tion. These data indicate that the localization of TgM-A is
directed by two precise residues rather than by the overall
positive charge of the tail domain.
DISCUSSION
A Family of Apicomplexan Myosins Presenting
Divergent Structural Features
T. gondii and P. falciparum are both obligate intracellular
parasites sharing a similar mechanism of host cell penetra-
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1395
tion. According to the capping model for host cell invasion,
a myosin is necessary to mediate the ATP-dependent actin-
based motility (Sibley et al., 1998). The molecular motor
mediating this process has not been identified so far. In the
present study, we have cloned and characterized two api-
complexan myosins that share a high degree of homology
with the previously identified class XIV of myosins (Heint-
zelman and Schwartzman, 1997). With a molecular mass
ranging between 90 and 93 kDa, these myosins are the
smallest molecular motors identified so far. Each of the T.
gondii myosins exhibit either a glutamine or an asparagine at
the TEDS site, which is neither acidic nor phosphorylatable,
potentially implying either different structural requirements
for activation or a different post-translational modification
involved in the regulation of these myosins. We are explor-
ing the speculative hypothesis that a deamidase might con-
vert asparagine or glutamine into acidic residues at the
TEDS site. Among the ⬎100 known myosins, there are few
others making exception to the TEDS rule, such as two
Acetabularia class XIII myosins, myosin IB of Entamoeba his-
tolytica (Vargas et al., 1997), and a Drosophila class III myosin
(Montell and Rubin, 1988). Like some T. gondii myosins, the
Drosophila myosin IA has an Asn (Strom Morgan et al., 1994).
Another striking feature of all apicomplexan myosins is the
presence of a serine instead of the highly conserved glycine
corresponding to Gly
699
in the chicken myosin II (Kinose et
al., 1996). This structural feature has only been reported for
a plant myosin (HaMyok3; D. Menzel, unpublished data)
and a Caenorhabditis elegans class XII myosin (Baker and
Titus, 1997). The conserved glycine residue likely plays an
essential role in the conformational changes around the
actin- and nucleotide-binding sites, maybe functioning as a
pivot point for the lever arm. Mutation of this glycine into an
alanine residue dramatically alters the motor activity of the
skeletal muscle myosin and the Dictyostelium discoideum my-
osin II, inhibiting the velocity of actin filament movement by
100-fold (Kinose et al., 1996; Patterson and Spudich, 1996;
Patterson et al., 1997). Finally, class XIV myosins do not carry
classical IQ motifs for the binding of light chains but harbor
a conserved stretch of amino acids that might represent a
very divergent form of this motif.
Because of these important structural divergences, it was
crucial to assess experimentally whether the apicomplexan
myosins truly act as ATP-driven, actin-dependent motors.
Also, a requirement of the capping model of invasion is that
the motor involved has to grab onto F-actin and exert ATP-
dependent traction. The ability to express and purify recom-
binant myosins from T. gondii offers a unique opportunity to
undertake combined in vivo and in vitro structure–function
analyses of this novel class of myosins. Taking advantage of
the solubility of mycTgM-A⌬tail, we purified the recombi-
nant protein from parasites under native conditions. This
material was used in pilot experiments aiming at the bio-
chemical characterization of TgM-A. Indeed, F-actin sedi-
mentation assay showed that TgM-A binds actin in an ATP-
dependent manner. Preliminary transient ATPase kinetics
and motility experiments indicate further that TgM-A puri-
fied from parasites is fully functional (our unpublished re-
sults).
TgM-A Localizes at the Plasma Membrane via Its
Tail Domain
By using a reverse genetic approach and by generating a
tail-specific antibody, we have determined the localization
of the T. gondii myosin TgM-A. It distributed beneath the
Figure 8. Cytochalasin treatment did not influence the peripheral
localization of mycTgM-A and GFP䡠TgM-Atail. GFP䡠TgM-Atail did
not localize to the plasma membrane of HeLa cells. (A) MycTgM-A
(a and b) and GFP䡠TgM-Atail (c and d) localized at the periphery of
parasites after incubation in the presence (b and d) or absence (a and
c) of 10
g/ml cytochalasin D for 3 h, indicating that it did not
depend on an intact actin cytoskeleton. To optimally present the
host actin cytoskeleton and the peripheral staining in the parasites,
one confocal section was recorded in the ventral part of the fibro-
blasts where the actin stress fibers are most prominent, and another
confocal section was recorded through the middle of the parasites,
1
m higher in the cell. Only the overlay is shown here. (B) Contrary
to what was seen in T. gondii, GFP䡠TgM-Atail did not interact with
the plasma membrane of an animal cell (d) and had a distribution
similar to GFP (b). (a and c) Phase-contrast pictures corresponding
to b and d.
C. Hettmann et al.
Molecular Biology of the Cell1396
Figure 9. A dibasic motif in the tail of TgM-A is essential for the cell membrane localization of GFP-tail chimera. (A) The wild-type sequence
of the tail of TgM-A (wt) is shown aligned with the site-specific mutants (I–III) and the two deletion mutants (⌬14 and ⌬22). The mutated
residues are indicated in red. (B) Confocal microscopic analysis of parasites stably transformed with the respective mutant tail constructs
fused to GFP. Indirect immunofluorescence was performed using the anti-myc antibodies. (C) Anti-myc immunoblot analysis of recombinant
parasites expressing the different constructs presented in A.
Plasma Membrane Localization of TgM-A
Vol. 11, April 2000 1397
plasma membrane of tachyzoites, an ideal position to trans-
mit mechanical energy in forward motion and thus to propel
cell invasion. At this point, we have to mention that Heint-
zelman and Schwartzman (1999) recently reported similar
data concerning the association of TgM-A with the particu-
late fraction of T. gondii, but contrary to our results, they
documented a punctate localization of TgM-A near the api-
cal pole of tachyzoites, possibly associated with some intra-
cellular structure or organelle such as the rhoptries. The
discrepancy is probably related to our problems in detecting
peripheral localization after aldehyde fixation associated
with the spurious detection of a cross-reactive rhoptry anti-
gen.
TgM-D appears enriched in the peripheral region of the
parasite but not as sharply defined as for TgM-A. The loss of
membrane targeting of TgM-A and TgM-D resulting from
deletion of their tail supports the idea that this domain
mediates interaction with cargo and brings the myosin to its
site of action. Conversely, fusion of the TgM-A tail to GFP
confers membrane association to that otherwise soluble cy-
tosolic protein. These data strongly support that the tail
domain is the necessary and sufficient determinant mediat-
ing specific interactions of the myosin with integral or pe-
ripheral membrane constituents.
Expression of mycTgM-A Down-regulates the Level
of Endogenous TgM-A
MycTgM-A and GFP䡠TgM-Atail transgenes were well ex-
pressed in the parasites. Whereas the GFP chimera was
essentially associated with the periphery and the particulate
fraction, only approximately half of mycTgM-A behaved
that way, suggesting that the specific membrane “receptor”
is saturable. Intriguingly, drastic down-regulation of endog-
enous TgM-A was observed upon expression of recombi-
nant mycTgM-A. This phenomenon appeared to be associ-
ated with overexpression of the full TgM-A and at least
partially also of GFP䡠TgM-Atail but was not seen with my-
cTgM-A⌬tail. We hypothesize that the parasites autoregu-
late the level of TgM-A in accordance with the abundance of
a receptor present in limiting amounts at the cell periphery.
The difficulties encountered in the generation of recom-
binant parasites expressing TgM-D and the very low level
of protein produced suggest a similar phenomenon. The
high expression levels of TgM-D⌬tail contrast with the
low expression levels of GFP䡠TgM-Dtail and argue for a
regulatory effect of the tail domain. Analogously, the tail
of PfM-A seems to induce a comparable effect. It is con-
ceivable that the limited expression of TgM-D, GFP䡠TgM-
Dtail, and GFP䡠PfM-Atail are due to their inappropriate
presence in cells lacking either a stage- or species-specific
partner, respectively. The potential role of stage-specific
regulation is not well understood yet. Preliminary studies
on the developmental pattern of T. gondii myosin gene
expression suggest that TgM-A is constitutively expressed
in tachyzoites and bradyzoites, whereas TgM-D tran-
scripts accumulate predominantly in bradyzoites (F. Del-
bac, A. Sa¨nger, C. Toursel, S. Tomavo, and D. Soldati,
unpublished results).
Note that in all cases expression is limited, but we did
not notice deleterious effects on the morphology and pro-
liferation of parasites. This contrasts with observations
reported for the expression of other myosins. Overexpres-
sion of entire unconventional myosin myoIB from D. dis-
coideum is an example of deleterious effects leading to a
phenotype as severe as observed in null cells (Novak and
Titus, 1997). Similarly, the overexpression of MYO4 or
MYO2 in S. cerevisiae led to morphological abnormalities
(Haarer et al., 1994).
The exact nature of the interaction between TgM-A tail
and the cell membrane is not elucidated yet; however, in
addition to the down-regulation phenomenon discussed
above, the following evidence further points toward a par-
asite-specific proteinaceous receptor. First, the fractionation
experiments with mycTgM-A as well as GFP䡠TgM-Atail ar-
gue for a very strong interaction of the tail with the constit-
uents of the plasma membrane. Second, the tails of TgM-A
and PfM-A have similar overall charges but markedly differ
in their association with the periphery. Third, the mutagen-
esis experiments indicate that two specific residues and not
simply the overall charge of the tail domain are important
for localization, rendering it unlikely that it interacts non-
specifically with phospholipids. This is corroborated by the
fact that the GFP䡠TgM-Atail had an exclusively cytoplasmic
distribution in HeLa cells. Finally, after aldehyde fixation,
the failure to detect the tail antigen specifically when TgM-A
is at the plasma membrane suggests that it is engaged in
strong interactions with a protein component.
Is TgM-A or TgM-D Powering Gliding Locomotion?
To accommodate the capping model for invasion, the myo-
sin shall lie beneath the plasma membrane and interact with
transmembrane proteins exposed at the cell surface at the
time of invasion. The motility shall then be driven by the
concerted redistribution of these transmembrane proteins
toward the posterior pole of the parasite. The TRAP protein
(trombospondin-related adhesive protein) expressed in the
sporozoites of Plasmodium species and its homologue MIC2
in T. gondii (Wan et al., 1997) are exported at the surface of
the parasite and relocalize to the posterior pole during in-
vasion (Carruthers et al., 1999). This protein is an excellent
candidate to interact directly or indirectly with the actomy-
osin system. Disruption of the TRAP gene in Plasmodium
berghei demonstrates that this protein is necessary not only
for sporozoite infection of the mosquito salivary glands and
the rat liver but also for gliding motility of the sporozoites in
vitro (Sultan et al., 1997). The TgMIC2 C-terminal domain
has recently been shown to functionally complement TRAP
mutants lacking their C-terminal domain in P. berghei
(Kappe et al., 1999). Although MIC2 constitutes an attractive
partner for TgM-A during invasion, it is only transiently
present at the surface of the parasite during host cell pene-
tration and therefore is unlikely to serve as receptor for
TgM-A at the plasma membrane during the intracellular
replicative phase of the life cycle (Carruthers and Sibley,
1997). It is conceivable that an abundant protein serves as a
docking partner, ideally positioning the myosin close to its
later site of action. Once at the plasma membrane, the my-
osin could respond immediately to an activation signal and
trigger invasion by establishing a connection with proteins
such as MIC2.
A recent study provides evidence for a myosin II respon-
sible for the capping of surface receptors in E. histolytica
(Arhets et al., 1998). In light of such a finding, it is rather
surprising that no conventional myosin of type II has been
C. Hettmann et al.
Molecular Biology of the Cell1398
identified in Apicomplexa so far. In comparison, despite its
simplicity, even S. cerevisiae relies on the function of five
myosins from three distinct classes (Brown, 1997). Our most
recent studies revealed the presence of a fifth myosin of class
XIV in T. gondii (our unpublished results). Nevertheless,
because our screening is not exhaustive, we cannot yet ex-
clude the presence of other classes of myosins in T. gondii.
The genome sequencing project for P. falciparum is still in
progress, and the current status reveals that at least two
additional myosin genes are present in this parasite, both of
which appear to be closest to class XIV. Although not yet
confirmed by functional data, TgM-A is definitely guilty by
localization. Besides unraveling its potentially crucial role in
invasion, the investigation of the biochemical details of en-
zymatic and motile properties of this very divergent class of
unconventional myosins holds the promise of shedding
light on the fundamental requirements of myosin function.
Note added in proof. A fragment corresponding to the complete
PfM-A cDNA reported here was previously described as Pfmyo-1
(Pinder et al., 1998). We named all the class XIV myosins according
to the nomenclature introduced by Heintzelman and Schwartzman
(1997). To minimize the confusion, Pinder and colleagues now
kindly agree to rename Pfmyo-1 Pfmyo-A.
ACKNOWLEDGMENTS
We are indebted to C. Kistler for assistance and expertise in the actin
sedimentation assays and to Dr. J. Ajioka for assistance in the
screening of the T. gondii genomic libraries. We are very grateful to
Dr. J.F. Dubremetz for providing the biotinylated anti-SAG1 anti-
body and to Dr. D. Chakabarti for kindly and promptly providing
us with the P. falciparum cDNA clone Pf1550C. Dr. D Lawson at the
Sanger Center was instrumental in completing the PfM-A gene
sequence. The PfM-B and -C coding sequences were assembled from
sequence data of chromosomes 5 and 13 obtained from the Sanger
Center web site (http://www.sanger.ac.uk/Projects/P_falcipa-
rum/). Sequencing of P. falciparum chromosomes 5 and 13 was
accomplished as part of the Malaria Genome Project with support
by The Wellcome Trust. We thank Dr. J. Haseloff for sending us the
modified GFP (mgfp-5ER). This work was supported by Deutsche
Forschungsgemeinschaft grants SO 366/1-1 and SO366/1-2. E.S.
was supported by Deutsche Forschungsgemeinschaft grant SFB 352
(to T.S.).
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