School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK
Correspondence
Jorge Tovar
j.tovar{at}rhul.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this paper is AF513821.
Present address: School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Present address: Genetics and Molecular Biology Department, CINVESTAV, IPN, Zacatenco, 07360 Mexico DF, Mexico.
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INTRODUCTION |
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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 mitochondrial-like targeting presequence at their amino terminus. The 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.
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METHODS |
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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 http://www.sanger.ac.uk/Projects/E_histolytica/. 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 5' and 3' untranslated regions of the putative E. histolytica cpn10 homologue (Ehcpn10_280F, 5'-CCA CAA CCA ATT TCA ACA CG-3'; Ehcpn10_604R, 5'-AAA GAA GAG GAA TAA ACG AAT TTT ATG-3') 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 directionally 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 (5'-aga aga GGT ACC CCA CAA CCA ATT TCA ACA CG-3') and Ehcpn10_EcoRV-R (5'-tct tct GAT ATC GAT TTT TGC AAA AAT GTC-3') (stuffer regions are indicated in lower case, restriction sites in italics, and perfect matches in roman capitals). The resulting construct pJT1-Ehcpn10 (Fig. 1a
) was sequenced as described above.
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RT-PCR studies on heat-shocked and control cells.
To test for expression, primers Ehcpn10_startF (5'-ATG GCA AAA ATT AAA CCA ACT GGA GAC ATG GTT TTA GTC C-3') and Ehcpn10_stopR (5'-TTA TTC GAT TTT TGC AAA AAT GTC ATG TTG TTT TAA TAA AG-3') were used for cpn10 amplification. In addition, cpn60- and actin-specific primers were used as controls (Eh_partCpn60281F, 5'- ATG GGA CAA CAA CAG CAA CA -3', and Eh_partCpn60544R, 5'- CAA CAG CAC CAT CTC TTC CA -3'; and Eh_actF, 5'- ATG GGA GAC GAA GAA GTT CA -3', and Eh_actR, 5'- AAG CAT TTT CTG TGG ACA AT -3'). 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 heat-shock experiments, E. histolytica trophozoites were incubated for 1 h at 42 °C (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 (http://www.expasy.org/spdbv/; 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.nih.gov/entrez/. 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 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).
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RESULTS |
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DISCUSSION |
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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 (http://www.sanger.ac.uk/Projects/E_histolytica) identified a gene encoding a putative homologue of the Cpn60 co-chaperonin Cpn10. Typical E. histolytica promoter elements seem to be present in the 5' 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 °C, 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 blotting.
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 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
).
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.
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
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.
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
; www.mbl.edu/Giardia) 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 evolution.
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 ironsulphur 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
).
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ACKNOWLEDGEMENTS |
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Received 25 March 2005;
revised 26 May 2005;
accepted 14 June 2005.
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