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Microbiology (2005), 151, 3107–3115
DOI 10.1099/mic.0.28068-0
Characterization of chaperonin 10 (Cpn10) from
the intestinal human pathogen Entamoeba
Mark van der Giezen,3 Gloria León-Avila4 and Jorge Tovar
Jorge Tovar
[email protected]
Received 25 March 2005
26 May 2005
Accepted 14 June 2005
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey
TW20 0EX, UK
Entamoeba histolytica is the causative agent of amoebiasis, a poverty-related disease that kills
an estimated 100 000 people each year. E. histolytica does not contain ‘standard mitochondria’,
but harbours mitochondrial remnant organelles called mitosomes. These organelles are
characterized by the presence of mitochondrial chaperonin Cpn60, but little else is known about the
functions and molecular composition of mitosomes. In this study, a gene encoding molecular
chaperonin Cpn10 – the functional partner of Cpn60 – was cloned, and its structure and
expression were characterized, as well as the cellular localization of its encoded protein. The 59
untranslated region of the gene contains all of the structural promoter elements required for
transcription in this organism. The amoebic Cpn10, like Cpn60, is not significantly upregulated
upon heat-shock treatment. Computer-assisted protein modelling, and specific antibodies against
Cpn10 and Cpn60, suggest that both proteins interact with each other, and that they function
in the same intracellular compartment. Thus, E. histolytica appears to have retained at least two of the
key molecular components required for the refolding of imported mitosomal proteins.
The human parasite Entamoeba histolytica was once considered as an example of a ‘primitive’ eukaryote since it lacks
typical features of ‘higher’ eukaryotes, such as mitochondria, peroxisomes, Golgi apparatus or rough endoplasmic
reticulum (Meza, 1992). Entamoeba, along with all other
amitochondriate eukaryotes, was thought to have diverged
from the main eukaryotic trunk before the mitochondrial
endosymbiont became established. Their putative primitive
nature appeared to be supported by rRNA phylogenies that
placed the microsporidia (e.g. Vairimorpha, Trachipleistophora, Encephalitozoon), diplomonads (e.g. Hexamita,
Spironucleus, Giardia) and parabasalids (e.g. Tritrichomonas,
Trichomonas) as the deepest, most ancestral branches of the
evolutionary tree (Sogin, 1989). However, the proposed premitochondrial nature of Entamoeba did not receive support
from rRNA phylogenetic reconstructions, as it branched
after well-established mitochondrial groups (Sogin, 1991).
This placement suggested the possibility that Entamoeba
might have mitochondrial ancestors.
3Present address: School of Biological and Chemical Sciences, Queen
Mary, University of London, Mile End Road, London E1 4NS, UK.
4Present address: Genetics and Molecular Biology Department,
CINVESTAV, IPN, Zacatenco, 07360 Mexico DF, Mexico.
Abbreviations: ML, maximum likelihood; UTR, untranslated region.
The GenBank/EMBL/DDBJ accession number for the nucleotide
sequence reported in this paper is AF513821.
0002-8068 G 2005 SGM
Over the past few years, several genes of mitochondrial ancestry have been identified in the genomes of
‘amitochondrial’ eukaryotes (reviewed by Embley & Hirt,
1998), and the reliability of rRNA phylogenies to resolve
early evolutionary events has been brought into question
(Gribaldo & Philippe, 2002). The discovery of mitochondrial genes in amitochondrial protists led to the subsequent
identification of mitosomes, intracellular compartments
housing mitochondrial proteins that are thought to represent mitochondrial remnants. Originally discovered in E.
histolytica (Mai et al., 1999; Tovar et al., 1999), mitochondrial remnant organelles have also been identified in
Trachipleistophora hominis (Williams et al., 2002), Giardia
intestinalis (Tovar et al., 2003) and Cryptosporidium parvum
(Riordan et al., 2003), and it is becoming apparent that
their distribution within the protist kingdom is widespread.
Thus, it is clear that the absence of classic mitochondria in
these organisms is a secondarily derived condition rather
than a primitive trait (van der Giezen et al., 2005).
Because of their recent discovery, little is known about the
functions and metabolic composition of mitosomes. Only
three genes encoding mitochondrial proteins have been
described in E. histolytica, i.e. pyridine nucleotide transhydrogenase (PNT), molecular chaperonin 60 (Cpn60)
and mitochondrial heat-shock protein 70 (mHsp70)
(Bakatselou et al., 2000; Clark & Roger, 1995; Tovar et al.,
1999). All these proteins seem to contain a mitochondriallike targeting presequence at their amino terminus. The
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M. van der Giezen, G. León-Avila and J. Tovar
putative targeting signal on the Cpn60 has been shown to
be required for targeting the protein into the E. histolytica
mitosome. When the targeting sequence was deleted, the
protein accumulated in the cytosol, a mutant phenotype that could be reversed by the addition of a functional
mitochondrial targeting signal from the Trypanosoma cruzi
mHsp70 protein (Tovar et al., 1999).
The availability of sequencing data from the recently completed E. histolytica genome project (Loftus et al., 2005)
allows the search for additional remnant mitochondrial
genes/proteins, and may provide additional information as
to the metabolic capacity and protein composition of the
mitosome. Here, we report on the identification and structural characterization of a gene encoding the molecular
chaperonin Cpn10 – the functional partner of Cpn60 (Hartl,
1996). We find that the upstream region of the gene contains
the promoter elements required for transcription in E.
histolytica, and that – similar to its functional partner –
transcription of cpn10 is not significantly upregulated by
heat shock. Amoebal Cpn10 is compartmentalized, and, like
many other Cpn10 proteins, it is targeted into the organelle
via a mechanism that does not require an amino-terminal
targeting presequence.
Cloning and sequencing of the E. histolytica cpn10 gene.
The human mitochondrial Cpn10 protein sequence (GenBank accession no. Q04984) was used to search the E. histolytica genome
sequence database at
Clones Ent141h02.p1c, Ent066d09.q1c, Ent066d09.p1c and
Ent091d08.q1c contained sequences similar to the human mitochondrial cpn10 gene sequence. Based on an alignment of these
clones, oligonucleotide primers complementary to the 59 and 39 untranslated regions of the putative E. histolytica cpn10 homologue
(Ehcpn10_280F, 59-CCA CAA CCA ATT TCA ACA CG-39;
Ehcpn10_604R, 59-AAA GAA GAG GAA TAA ACG AAT TTT ATG39) were synthesized and used for PCR amplification; E. histolytica
genomic DNA was used as a template. Amplified DNA products
were purified, cloned into the pGEM-T-Easy vector (Promega), and
sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit on
an Applied Biosystems 377-96 DNA Sequencer.
Construction of the expression vector harbouring E. histolytica cpn10. The putative E. histolytica cpn10 gene was cloned direc-
tionally into the KpnI and EcoRV restriction sites of the Entamoeba
expression vector pJT1 (Fig. 1a). pJT1 was obtained by removing
the cpn60 gene from construct A (Tovar et al., 1999) – a derivative
of expression vector pEhNEO/CAT (Hamann et al., 1995). KpnI and
EcoRV restriction sites were added at the termini of Ehcpn10 by
means of PCR using the primers Ehcpn10_KpnI-F (59-aga aga GGT
ACC CCA CAA CCA ATT TCA ACA CG-39) and Ehcpn10_EcoRVR (59-tct tct GAT ATC GAT TTT TGC AAA AAT GTC-39) (stuffer
regions are indicated in lower case, restriction sites in italics, and
perfect matches in roman capitals). The resulting construct pJT1Ehcpn10 (Fig. 1a) was sequenced as described above.
Parasite transfection. E. histolytica trophozoites were detached
Micro-organism cultivation, and DNA manipulations. E. histo-
lytica HM-1 : IMSS clone 9 was maintained axenically by subculture
in YI-S medium with 15 % adult bovine serum, as described by
Clark & Diamond (2002). E. histolytica genomic DNA was isolated using cetyltrimethylammonium bromide (CTAB), according to
Clark (1992). Escherichia coli strain DH5a was grown in Luria–
Bertani (LB) medium (with 100 mg ampicillin ml21) at 37 uC.
Standard recombinant DNA techniques were used, as described elsewhere (Sambrook et al., 1989).
from culture tubes by chilling on ice, and collected by centrifugation
at 500 g at 4 uC for 10 min. The cell pellet was washed twice
with ice-cold PBS, and once in cytomix buffer consisting of
10 mM K2HPO4/KH2PO4 (pH 7?6), 120 mM KCl, 0?15 mM CaCl2,
25 mM HEPES, 2 mM EGTA and 5 mM MgCl2 (Nickel & Tannich,
1994). Trophozoites (16107) were resuspended in 0?8 ml cytomix,
and supplemented with 4 mM ATP, 10 mM glutathione and
100 mg pJT1-Ehcpn10 DNA in an electroporation cuvette (4 mm
gap). Cells were electroporated twice using a Bio-Rad Gene Pulser II
with pulse controller and capacitance extender, using 3000 V cm21
Fig. 1. Structure of recombinant constructs and of the 59-flanking region of the E. histolytica cpn10 gene. (a) pJT1 contains a
c-myc epitope tag (filled box) in-frame with the EcoRV restriction site. The actin and lectin transcriptional regulatory elements
present in the plasmid are indicated (see Methods; Hamann et al., 1995). Cloning of the E. histolytica cpn10 gene into pJT1
resulted in an in-frame fusion construct tagged at its carboxy terminus (pJT1-Ehcpn10) that was used to generate transgenic
parasite lines. (b) The three typical upstream regulatory elements are depicted as shown by Purdy et al. (1996): the putative
initiator element, double underlined; the ‘GAAC’ element, grey box; and the putative TATA element, boxed.
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Microbiology 151
Entamoeba histolytica Cpn10
and 25 mF. Electroporated cells were incubated on ice for 10 min,
after which 13 ml fresh culture medium was added. After 48 h
incubation at 37 uC, G418 (6 mg ml21) was added to select for
100 times using SEQBOOT from the PHYLIP 3.6 package (Felsenstein,
1993). The ML program was run with these resampled data. A
majority-rule consensus tree was obtained from the resulting 100
trees using CONSENSE (PHYLIP).
RT-PCR studies on heat-shocked and control cells. To test
for expression, primers Ehcpn10_startF (59-ATG GCA AAA ATT
TTG TTT TAA TAA AG-39) were used for cpn10 amplification. In
addition, cpn60- and actin-specific primers were used as controls
(Eh_partCpn60281F, 59- ATG GGA CAA CAA CAG CAA CA -39,
and Eh_partCpn60544R, 59- CAA CAG CAC CAT CTC TTC CA
-39; and Eh_actF, 59- ATG GGA GAC GAA GAA GTT CA -39, and
Eh_actR, 59- AAG CAT TTT CTG TGG ACA AT -39). Total RNA
was isolated using TriPure (Roche). DNase-treated total RNA was
used as a template; the RNA was tested for DNA contamination by
means of a standard PCR reaction using the same primers. For heatshock experiments, E. histolytica trophozoites were incubated for 1 h
at 42 uC (Mai et al., 1999), after which total RNA was isolated as
described above. Ethidium-bromide-stained gels were digitized using
a GeneFlash gel documentation system (SynGene), and band intensities were quantified by densitometry using ImageQuant of the IQ
Solutions software package (Amersham Biosciences).
Fractionation and Western blotting. E. histolytica trophozoite
extracts were fractionated as described previously for G. intestinalis
(Tovar et al., 2003), followed by SDS-PAGE and Western blotting
using a semidry electroblotter. Blots were stained using heterologous
antiserum (1 : 50) against human Cpn10 (Santa Cruz Biotechnology), or homologous antiserum (1 : 1000) against E. histolytica
Cpn60 (Tovar et al., 1999), followed by secondary anti-rabbit IgG
antibodies conjugated with horseradish peroxidase, and visualized
by chemiluminescence.
Three-dimensional modelling. The E. histolytica Cpn10 putative
three-dimensional structure was deduced using the conceptually
translated Ehcpn10 sequence. The previously solved crystal structure
of Mycobacterium tuberculosis Cpn10 (PDB accession no. 1HX5;
Taneja & Mande, 2002) was used as a template. The E. histolytica
Cpn10 sequence was aligned to the M. tuberculosis Cpn10 sequence
using DeepView v3.7 sp8 (; Guex &
Peitsch, 1997), and manually improved based on an independent
CLUSTAL W alignment (Thompson et al., 1994).
Phylogenetic analyses. Protein database searches using the
deduced E. histolytica Cpn10 amino acid sequence were performed
at the National Center for Biomedical Information (NCBI) using
BLAST (Altschul et al., 1990). Several Cpn10 homologues were
retrieved through the NCBI Entrez server at http://www.ncbi.nlm. The Cpn10 sequence of E. histolytica was aligned to
sequences from 21 taxa using CLUSTAL_X (Thompson et al., 1997).
The alignment was further edited visually with the use of BioEdit
(Hall, 1999), and regions of ambiguous alignment, and residues with
gaps, were excluded from the analysis, leaving a final dataset of 21
taxa and 69 amino acid positions. Bayesian searches of treespace
were performed with the program MRBAYES (Huelsenbeck &
Ronquist, 2001), using the JTT-f amino acid substitution matrix
with four variable gamma rate corrections. Two hundred thousand
Monte Carlo Markov Chain generations were performed, with trees
sampled every 100 generations. For compilation of the Bayesian consensus topologies, a ‘burn-in’ of 500 trees was used, leaving 1500
trees to estimate separate 50 % majority-rule consensus trees.
Maximum-likelihood (ML) analysis was performed using PHYML
(Guindon & Gascuel, 2003) with a mixed four-category discrete
gamma model of rate heterogeneity (i.e. substitution model+
gamma). The JTT-f substitution model adjusted for character frequencies was used in the ML analysis. Protein data were resampled
Identification, isolation and characterization of
the E. histolytica gene encoding Cpn10
Searches of preliminary data generated by the E. histolytica
genome project at the Sanger Institute revealed several
clones with sequence similarity to the human Cpn10 protein
sequence. PCR primers based on these clones, when used in
PCR reactions on E. histolytica genomic DNA as template,
amplified a 324 bp fragment, which was purified, cloned
and sequenced. The E. histolytica cpn10 nucleotide sequence
has been submitted to GenBank under accession number
AF513821, and is identical to the nucleotide coding sequence for the conceptually translated protein entry EAL51277
(Loftus et al., 2005). A BLAST search of the entire published
genome indicated that there is only a single copy of cpn10
present. The 59 untranslated region of the amoebal cpn10
gene contains distinct putative promoter elements reported
to be typical for E. histolytica (Purdy et al., 1996). The
initiator region, and the putative TATA and GAAC boxes,
are all present within the first 30 bases upstream of the start
codon (Fig. 1b), suggesting that the gene is functional. The
E. histolytica cpn10 gene encodes a protein of 87 aa, with a
predicted molecular mass of 9?7 kDa, and a theoretical
isoelectric point of 9?3. The G+C content is 31 mol% for
the coding region, 21 mol% for the 59 untranslated region
(UTR), and 25 mol% for the 39 UTR. The mean G+C
content of E. histolytica genes is 32 mol% based on 241 genes
analysed (Nakamura et al., 2000). No introns were present
in the ORF. The E. histolytica chaperonin contains Pfam
domain PF00166, which is typical for this protein family. To
test whether the protein assumes a normal three-dimensional conformation, its amino acid sequence was modelled
on the solved Cpn10 protein structure from M. tuberculosis
(Taneja & Mande, 2002). The identity between the two
proteins is 26 %, indicating that they belong to the same fold
(the GroES fold; Taneja & Mande, 1999). The overall
topologies of both the monomeric (Fig. 2a) and the heptameric (Fig. 2b) models are quite similar to the solved
M. tuberculosis structure, suggesting that the E. histolytica
protein is indeed involved in a chaperonin function. The
alignment (Fig. 2c) indicates that deletions in the E.
histolytica protein occur in connecting loops only, and
not in the b strands of this b-barrel protein, further
supporting the validity of the model.
Effect of heat shock on expression of E.
histolytica Cpn10
To check if the E. histolytica cpn10 gene is expressed, we
performed RT-PCR experiments as described in Methods.
Using total RNA isolated from E. histolytica trophozoites, a
band of 0?3 kb was obtained, in agreement with the size
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M. van der Giezen, G. León-Avila and J. Tovar
Fig. 2. (a, b) Three-dimensional models of E. histolytica Cpn10, drawn using MOLMOL (Koradi et al., 1996). The E. histolytica
Cpn10 putative three-dimensional monomeric (a) and heptameric (b) structures were deduced using the conceptually
translated Ehcpn10 sequence. The previously solved crystal structure of M. tuberculosis Cpn10 (PDB accession no. 1HX5;
Taneja & Mande, 2002) was used as a template. (c) The E. histolytica Cpn10 sequence was aligned with the M. tuberculosis
Cpn10 sequence using DeepView v3.7 sp8 (; Guex & Peitsch, 1997), and manually improved
based on an independent CLUSTAL W alignment (Thompson et al., 1994). Predicted b strands, and the Cpn60-binding
hydrophobic tripeptide, are indicated.
predicted from the gene sequence (Fig. 3, lane 1). Since, in
several other organisms, heat-shock proteins can be induced
upon incubation at elevated temperatures, a semi-quantitative comparison of amplified DNAs from control and
heat-shocked parasites (incubated at 42 uC for 1 h; Mai et al.,
1999) was carried out. No major increase of amplified cpn10
(118 %, n=3) and cpn60 (116 %, n=3) DNA was observed
upon incubation at 42 uC using this method (Fig. 3), suggesting that these genes are not subjected to heat-shock
regulation in E. histolytica. This observation is in contrast to
the reported heat-shock inducibility of the E. histolytica
cpn60 gene at 42 uC (Mai et al., 1999), but in agreement with
our previous findings using Northern and Western dot
blotting (Tovar et al., 1999).
contains a functional targeting presequence that helps to
target the protein into the mitochondrial remnant organelles. Given that Cpn10 is the functional partner of Cpn60,
we hypothesized that E. histolytica Cpn10 might also be
targeted into mitosomes. Alignment of amoebal Cpn10 with
homologues from bacteria and other eukaryotes failed to
identify a putative targeting presequence (Fig. 4), suggesting
that, if targeted into mitosomes, the amoebal protein may be
Cellular localization of E. histolytica Cpn10
Proteins destined for the mitochondrial lumen usually contain characteristic amino-terminal targeting presequences
that are proteolytically removed upon import into the
organelle (Pfanner & Truscott, 2002). E. histolytica Cpn60
Fig. 3. RT-PCR analysis of E. histolytica cpn10, using RNA
from cells maintained at 37 6C (lane 1) and RNA from cells
heat-shocked for 1 h at 42 6C (lane 2). Lanes 3–6, templates
as before, but using either cpn60- or actin-specific primers.
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Microbiology 151
Entamoeba histolytica Cpn10
Fig. 4. Analysis of the amino-terminal region of the E. histolytica Cpn10 protein with Cpn10 proteins from the eukaryotes
Trichomonas vaginalis, Leishmania donovani, Schizosaccharomyces pombe, Mus musculus, and bacterial homologues
from Yersinia pestis and Brucella abortus. Identical aligned residues are indicated in dark-grey boxes, while conserved residues according to the PAM250 matrix (Schwartz & Dayhoff,
1978) are indicated by light-grey boxes.
imported into the organelle by a targeting mechanism that
does not require amino-terminal extensions. This would not
be unprecedented, as several other mitochondrial Cpn10
proteins that lack an amino-terminal extension have been
reported (Hartman et al., 1992).
To investigate the cellular localization of E. histolytica
Cpn10, cellular fractions were prepared, blotted onto a solid
support, and probed with a heterologous polyclonal antibody raised against the human Cpn10 homologue (Santa
Cruz Biotechnology). Using this antibody at a low dilution
(1 : 50), a very faint band of the predicted size was observed
in the mixed membrane fraction (not shown). To boost
the signal, transgenic E. histolytica lines harbouring pJT1Ehcpn10 (Fig. 1a) were generated. These parasites overexpress the amoebal Cpn10 antigen tagged with a c-myc
epitope. Immunoblotting of the overexpressing parasite
cellular fractions showed cross-reactivity of the antibody
with a protein enriched in the mixed-membrane fraction
(Fig. 5). The apparent molecular mass of 11 kDa for Cpn10
is identical to the calculated molecular mass of the predicted epitope-tagged gene product. When the antiserum
raised against the E. histolytica Cpn60 (Tovar et al., 1999)
was used, Cpn60 was observed in the cell-free extract and in
the mitosomal fraction, but not in the cytosolic fraction
(Fig. 5). Detection of Cpn10 in the cytosolic fraction is likely
to be the result of overexpression and limited organelle
import capacity. Despite repeated attempts, the use of a
monoclonal anti-tag antibody failed to detect the overexpressed protein in Western blotting and immunomicroscopy experiments. One possible explanation for this
observation would be that the tag is not sufficiently exposed
on the surface of the heptameric structure for efficient
interaction with the antibody; alternatively, codon usage
may prevent efficient biosynthesis of the tag in E. histolytica.
Either way, the high level of Cpn10 overexpression, and its
observed distribution in transgenic parasites (Fig. 5), makes
it unsuitable for the localization of the native antigen by laser
scanning confocal microscopy (León-Avila & Tovar, 2004).
Fig. 5. Localization of the E. histolytica Cpn10 protein in cellular fractions. E. histolytica cells were lysed, and subsequently
fractionated into a cytoplasmic supernatant and an organellar
pellet. After electrophoresis and Western blotting, membranes
were immunolabelled using anti-human Cpn10 antiserum in a
1 : 50 dilution, or anti-E. histolytica Cpn60 antiserum (Tovar
et al., 1999) in a 1 : 1000 dilution. lys., total lysate; cyt., cytoplasmic fraction; mit., organellar pellet. Each lane contains
10 mg protein.
Phylogenetic analyses of the E. histolytica
Bayesian and ML analyses of E. histolytica Cpn10 revealed
that phylogenetic relations are poorly resolved (Fig. 6).
Although major eukaryotic clades receive proper support,
their branching order is unresolved. The placement of E.
histolytica at the base of eukaryotes is probably a reflection
of limited sampling, since no ‘deep-branching’ eukaryotes
other than Trichomonas vaginalis are present. Nonetheless,
the E. histolytica Cpn10 sequence is part of the large eukaryotic clade, consistent with the Cpn60 phylogeny (Roger
et al., 1998; Horner & Embley, 2001). The clustering of
T. vaginalis with bacteria does not receive statistical support, and is therefore unresolved. The low phylogenetic
signal of small proteins such as Cpn10 has been suggested
previously (Fast et al., 2002).
Until recently, the origin of eukaryotes was explained by a
simple model that assumed the gradual acquisition of typical eukaryotic traits that led from a simple bacterial-like
cellular architecture to a more complex eukaryotic one
(Margulis, 1970). This view was based on the existence of
contemporary microbial eukaryotes that contain a nucleus,
but lack double-membrane-bounded organelles, such as
mitochondria and chloroplasts. The identification of genes
encoding putative mitochondrial proteins (e.g. Cpn60,
mtHsp70) in the genomes of several amitochondrial eukaryotes (Bui et al., 1996; Clark & Roger, 1995; Germot et al.,
1996; Hashimoto et al., 1998; Horner et al., 1996; Roger et al.,
1996, 1998), and the cellular localization of their encoded
proteins by immunomicroscopy, led to the discovery of
organelles of mitochondrial ancestry – collectively known as
mitosomes (Mai et al., 1999; Riordan et al., 2003; Tovar et al.,
1999, 2003; Williams et al., 2002) – and to the demonstration that trichomonad hydrogenosomes – organelles discovered in the early 1970s – shared a common ancestry with
mitochondria (reviewed by van der Giezen et al., 2005).
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M. van der Giezen, G. León-Avila and J. Tovar
Fig. 6. Phylogenetic analyses of the E. histolytica Cpn10 protein. An unrooted ML phylogenetic tree of 21 protein sequences
is shown. The E. histolytica Cpn10 sequence is a basal eukaryotic branch with low support. Support values are shown at the
nodes: left, bootstrap values above 50 % as determined using PHYML (Guindon & Gascuel, 2003); right, posterior probabilities
above 0?75 as determined by MRBAYES (Huelsenbeck & Ronquist, 2001). Note that for E. histolytica, lower support values are
shown in order to indicate the level of support for this branch.
The exact protein complement of mitochondrion-related
organelles remains to be determined. In E. histolytica, candidates to make up the mitosome are still limited: the
molecular chaperonins Cpn60 (Clark & Roger, 1995; Tovar
et al., 1999) and Hsp70 (Bakatselou et al., 2000), pyridine
nucleotide transhydrogenase (PNT) (Clark & Roger, 1995),
and possibly IscU and IscS (Ali et al., 2004; van der Giezen
et al., 2004). Only Cpn60 has actually been shown to reside
inside the mitosome, while evidence for the other proteins
is circumstantial. Screening of the E. histolytica genome
( identified
a gene encoding a putative homologue of the Cpn60 cochaperonin Cpn10. Typical E. histolytica promoter elements
seem to be present in the 59 UTR. E. histolytica is quite
unique in having three core promoter elements: apart from
the standard TATA-like sequence and a putative initiator,
there is a third GAAC-element of unknown function that
is also present in the upstream region of cpn10. Heat shock
did not induce significantly higher expression levels of
either cpn10 or cpn60 (Fig. 3). This is in contrast to earlier
observations by Mai et al. (1999), who reported increased
production of Cpn60 in parasites heat-shocked at 42 uC,
but it is in agreement with findings by Tovar et al.
(1999), who did not detect a significant increase in Cpn60
expression upon heat shock using Northern and Western
Co-localization of Cpn10 with Cpn60 is suggested by
immunolabelling of cellular fractions, a distribution consistent with their functional partnership in protein folding
(Hartl, 1996). Cpn60 and Cpn10 form large multimeric
protein complexes consisting of two stacked heptameric
rings of Cpn60, and a smaller single heptameric ring of
Cpn10 subunits (Hartl, 1996). A hydrophobic tripeptide
demonstrated to be involved in the interaction between
Cpn10 and Cpn60 (Richardson et al., 2001) is conserved in
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Microbiology 151
Entamoeba histolytica Cpn10
the E. histolytica Cpn10 (Fig. 2c). The large Cpn60-barrel
provides an enclosed folding compartment, but the association of Cpn10 with this Cpn60 structure induces conformational changes that actually allow unfolded proteins
to bind. The presence of the interacting partner of the E.
histolytica Cpn60 (which is known to be localized inside the
mitosomes) suggests that chaperonin-assisted protein folding does occur in this organelle. Since chaperonin-assisted
protein folding is an ATP-dependent process, our data
suggest that ATP is either imported into the mitosomes or
generated inside the organelle. Current evidence suggests
that the former process, and not the latter, operates in E.
histolytica mitosomes. An unusual member of the ADP/ATP
mitochondrial carrier family of transporters has been
recently identified and characterized in this organism
(Chan et al., 2005). In contrast to typical ADP/ATP carriers,
the E. histolytica carrier is not reliant on a membrane
potential for function, nor is it inhibited by classic inhibitors
such as carboxyatractyloside and bongkrekic acid. Although
most eukaryotes have many mitochondrial carriers, E.
histolytica contains only a single mitochondrial carrier in
its genome (Chan et al., 2005; Loftus et al., 2005). There is
no evidence to suggest that energy metabolism might be
compartmentalized in E. histolytica (Müller, 2000).
It is becoming apparent that the diversity of mitochondrial
remnants in various eukaryotic lineages is reflected in the
heterogeneity of their individual protein complements. E.
histolytica mitosomes, like T. vaginalis hydrogenosomes,
contain a typical mitochondrial protein refolding system,
i.e. Cpn60, Cpn10 and mtHsp70 (this work; Bakatselou et al.,
2000; Bui et al., 1996; Tovar et al., 1999). G. intestinalis
contains genes encoding homologues of chaperonin Cpn60
and mtHsp70 proteins (Arisue et al., 2002; Horner &
Embley, 2001; Morrison et al., 2001; Roger et al., 1998; Tovar
et al., 2003), but standard screening of the Giardia genome
database (McArthur et al., 2000;
provides little evidence for the presence of chaperonin
Cpn10. In contrast, the genome of the microsporidian
Encephalitozoon cuniculi does not appear to have genes
encoding Cpn60 or Cpn10, but has retained a mtHsp70
(Katinka et al., 2001). So, while these molecular chaperones
have been widely used as reliable tracers of mitochondria,
they have not necessarily been retained in all mitochondrion-related organelles. It is possible that, like many other
proteins found in the original mitochondrial endosymbiont, molecular chaperones may have been retargeted to a
different cell compartment, or lost during the course of
Although the evolutionary origins of Cpn10 could not be
unequivocally established due to insufficient phylogenetic
signal, the mitochondrial ancestry of Cpn60 is well documented (Horner & Embley, 2001; Viale & Arakaki, 1994;
Clark & Roger, 1995). Since Cpn10 and Cpn60 are
interacting partners, one might assume that both have a
similar, if not identical, ancestry. However, in order to
reliably trace this past, more phylogenetic signal than these
69 aa can provide is needed, as noted by Fast et al. (2002).
Major clades are supported by reasonably high bootstrap
support, but ‘deeper’ relationships remain without support.
Information from the first published genome sequence of
any amitochondrial eukaryote – that of the microsporidian
Encephalitozoon cuniculi (Katinka et al., 2001) – suggested
that the principal function of the then hypothetical microsporidian mitosome might be iron–sulphur cluster (Isc)
assembly, an essential function of mitochondria (Lill &
Muhlenhoff, 2005). Homologues of isc genes have now been
identified in the genomes of many other ‘amitochondrial’
eukaryotes, including Giardia, Cryptosporidium, Trichomonas and Entamoeba (Ali et al., 2004; LaGier et al., 2003;
Lill & Muhlenhoff, 2005; Tachezy et al., 2001; Tovar et al.,
2003; van der Giezen et al., 2004). Whilst the cellular localization of Isc proteins in Cryptosporidium and Entamoeba
remains to be unequivocally demonstrated, in Giardia and
Trichomonas, these proteins have been localized to mitosomes and hydrogenosomes, respectively (Sutak et al., 2004;
Tovar et al., 2003). The apparent ubiquity of Isc proteins
prompted the suggestion that Isc assembly might have
played an important role in the original endosymbiont that
gave rise to mitochondria (Embley et al., 2003; Tovar et al.,
2003; van der Giezen et al., 2005). Identifying the full metabolic capacity and protein complement of mitosomes may
lead to the identification of distinctive features that could
be exploited as antiparasitic drug targets – as has been elegantly demonstrated for the endosymbiosis-derived malarial apicoplast (Ralph et al., 2004).
Most mitochondrial matrix proteins contain amino-terminal presequences that are cleaved upon import into the
mitochondrion (Pfanner & Truscott, 2002). In contrast,
a number of mitochondrial proteins lack such a cleavable
presequence, but are nevertheless sorted to the mitochondrial matrix. The targeting information is thought to reside
within the amino-terminal part of the protein. No common
denominator seems to connect these proteins, since they are
involved in a variety of metabolic functions. Known mitochondrially targeted proteins without presequences are
bovine rhodanese, rat 3-oxoacyl-CoA thiolase, the b subunit
of human electron transfer flavoprotein, and yeast mitochondrial ribosomal protein YmL8 (Jarvis et al., 1995, and
references therein). Interestingly, several eukaryotic Cpn10
homologues have been identified (rat, bovine, human, yeast
and potato), and the absence of any amino-terminal targeting information seems to be a common phenomenon
(Legname et al., 1995). Our data suggest that the E. histolytica Cpn10 is another addition to this list, and indicate that
there must be some cryptic signal residing in this protein as
well, in order to sort it to the mitosomes.
The authors wish to thank Professor Richard Pickersgill for advice, and
Neil Sommerville for maintaining parasite cultures. This work was
supported by grants from the BBSRC (111/C13820) and the Wellcome
Trust (059845).
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