Entamoeba histolytica, a protist parasiteof the human intestine, is famous for its‘simple’ cell structure1–4 and ‘primitive’,‘bacterium-like’ metabolism4,5, and hasbeen regarded as an ancestral eukary-ote1,4. A prominent reason for this isthat E. histolytica contains no structurewith the morphological characteristics ofa mitochondrion3 nor the enzymes oftypical mitochondrial energy metab-olism1,6. However, two papers on anewly recognized structure, publishedalmost simultaneously, will dramaticallychange our views on E. histolytica. Tovaret al.7 and Mai et al.8 have reported theexistence and properties of a small or-ganelle, named, respectively, ‘mitosome’or ‘crypton’, a discovery also discussedrecently in Trends in Microbiology9. Theorganelle shares several biological char-acteristics with mitochondria and is likelyto have descended from a common an-cestor. The existence of such an or-ganelle, a mitochondrial ‘remnant,’ hasbeen predicted since the pioneering de-tection of two genes of putative mito-chondrial origin, encoding the proteinschaperonin 60 (cpn60) and pyridine nu-cleotide (NAD/NADP) transhydrogen-ase10 in E. histolytica. Now that this pu-tative organelle has been discovered, itopens up an important area of compara-tive cell biology and eukaryotic evolu-tion to experimental research.

The critical observation made byboth groups was the localization of oneof the products of these genes (cpn60)in a small structure in E. histolytica byconfocal fluorescence microscopy andcell fractionation7,8. Most cells containedonly one such organelle but a few cells,possibly those ready to divide, containedtwo, or occasionally three. Mai et al.8also used antibodies reacting with pro-teins of other subcellular compartments(nucleus, endoplasmic reticulum, Golgiapparatus and cytosol), which showeddistinctly different localizations in thecell. This supports the conclusion that theorganelle seen is an entity sui generis. Thefluorescent microscopic images (Fig. 5 inRef. 7 and Fig. 7a and 7b in Ref. 8) do notpermit the exact measurement of thesize of the organelle, but do suggest a di-ameter of 1–2 mm. The size of anamoeba is approximately 20 mm; thus,the organelle cannot represent .0.1%of the total cell volume.

The identification of these structuresas mitochondrial ‘remnants’ is based on

two findings: First, the protein detected,cpn60, is known to be restricted to mitochondria, hydrogenosomes andchloroplasts11. Sequence analysis andphylogenetic reconstruction placed theE. histolytica cpn60 robustly in a clade to-gether with its mitochondrial and hy-drogenosomal homologs9,12, indicatingan origin from a common ancestralgene. The most parsimonious expla-nation is that this ancestral gene was ac-quired through the endosymbiotic eventthat led to the establishment of the an-cestor of these organelles12, although al-ternative scenarios cannot be entirelyrejected.

Second, the fate of E. histolyticacpn60 is identical to that of its mito-chondrial and hydrogenosomal homo-logs7,8. The protein is encoded by nucleargenes and translation occurs on free ri-bosomes in the cytosol. The product isimported into the organelle post-trans-lationally, a process in which a processedN-terminal targeting peptide plays acrucial role. When cpn60 was encodedby constructs in which the codons forthe targeting peptide had been deleted,the protein did not enter the organellebut remained in the cytosol. If the tar-geting sequence was replaced by thetargeting sequence of a mitochondrialprotein of Trypanosoma cruzi, the pro-tein was found in the small organelle inE. histolytica7.

Cells of E. histolytica contain manymembrane-bound vacuoles and vesiclesof diverse sizes3. Thus, it should notcome as a surprise that, without the useof specific markers, a unique small bodyhad not been previously recognized.When the organelle is identified by im-munoelectron microscopy, it should bepossible to pinpoint it on previouslypublished electron micrographs. It is expected that, like mitochondria, the organelle will be enveloped by twomembranes.

It is not clear what else the organellecontains. A nuclear gene for a second‘mitochondrial-type’ protein, NAD/NADPtranshydrogenase, has also been foundin E. histolytica9, although it remains tobe established whether this protein getsimported into the organelle. A keymetabolic enzyme, pyruvate : ferredoxinoxidoreductase, has been localized to,among other structures, small cytoplas-mic bodies in E. histolytica13. However,the relationship of this structure to the

organelles discussed here remains to beelucidated. The small size of the or-ganelle indicates that it is not a major siteof metabolic flow, in contrast to mito-chondria, hydrogenosomes and peroxi-somes. Mitochondria usually occupy.10% of the cell volume, but can represent as much as one-third of it, eg.in hepatocytes, parabasalid hydro-genosomes are of the order of 10%.Peroxisomes with an active role in car-bohydrate catabolism or anabolism (gly-cosomes in kinetoplastids, plant peroxi-somes involved in photorespiration andplant glyoxysomes) also make a signifi-cant contribution to the total cell volume. Thus, one will have to look for functions that, while vital, are per-formed by enzymes in low copy num-ber. When the genome of this organismis sequenced (an event which cannot be too far off), a clearer image of thecomposition of the organelle shouldemerge, although its role will be harderto determine.

It is likely that identical organelles willbe detected in all Entamoeba spp andrelated parasitic amoebae (Endolimax,and possibly Jodamoeba). However,Dientamoeba fragilis, found in the humancolon, usually discussed together withother enteric amoebae, is an exception.Molecular data14 support earlier propo-sals that D. fragilis is a parabasalid, relatedto trichomonads15. The observation thatit harbors morphologically identifiablehydrogenosomes substantiates this tax-onomic position15.

The findings discussed here supportthe idea that Entamoeba spp and theirrelatives descended from an ancestorthat had experienced the ‘mitochondrialendosymbiotic event’, and their present‘simple’ cytological makeup is actually asecondary characteristic. In essence,they appear to have evolved by func-tional and morphological losses. Ofthese, perhaps the most dramatic is theloss of the major metabolic functions ofmitochondria and the retention of an,essentially cytosolic, extended glycoly-sis6,16. The reconstruction of this evolu-tionary history is hampered by the stillunclarified position of Entamoeba sppamong eukaryotes. They have no free-living relatives with similarly simple mor-phology and energy metabolism, andmolecular data on their position havebeen conflicting17. Based on diverse cri-teria, Cavalier-Smith18 suggested that

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A Mitochondrion in Entamoeba histolytica?M. Müller

their closest relatives are the slime-molds (eg. Dictyostelium and Physarum)and free-living ‘amitochondriate’ amoe-boflagellates called pelobionts (eg.Mastigamoeba and the giant amoeba,Pelomyxa). He placed all these organ-isms in the newly established sub-phylum, the Conosa of the phylumAmoebozoa. Sophisticated analyses ofsmall-subunit rRNA sequences supportthe relationship between entamoebidsand pelobionts and do not contradictthe relationship of these with the slime-molds17. A more detailed comparison ofall these organisms is likely to shed lighton the evolutionary history of entamoe-bids, which might just represent themost extreme examples of reductiveevolution among those eukaryotes thatdid not establish an intracellular parasiticmode of life.

Is the occurrence of homologous oranalogous organelles possible in otherorganisms? Likely candidates are thediplomonads, with Giardia and Hexamitaas the best-known parasitic representa-tives. These organisms do not containmitochondria or hydrogenosomes asorganelles of core metabolism, althougha gene with mitochondrial ancestry en-coding cpn60 has been detected in G. lamblia12. The free-living pelobiontsalso have small intracellular bodies thatcould be similar in nature to those foundin E. histolytica19.

The study of only a few unicellulareukaryotes revealed an unexpectedstructural and metabolic diversity com-patible with the eukaryotic phenotype.Among these are the ‘amitochondriate’protists, which showed that this pheno-type is not linked obligatorily to thepresence of a mitochondrion perform-ing oxidative phosphorylation6,16. In thepast two decades, the origin of such‘amitochondriate’ organisms has be-come a hotly debated topic of evolution-ary studies16,20,21. The view that ‘amito-chondriate’ protists are relics of theancestral, premitochondriate eukaryoticcell is now less popular. Mounting evi-dence indicates that all the ‘amitochon-driate’ eukaryotes studied to date arosefrom ancestors that experienced thesame endosymbiotic event that led to the establishment of the ancestral mitochondrion16,20. Perhaps the most studied examples are the parabasalids,primarily the trichomonads. Their charac-teristic metabolic organelle, the hy-drogenosome, is generally considered aderivative of the ancestral mitochondrialsymbiont22,23. Hydrogenosomes, possi-bly differing in their composition, havealso been detected in other protists and anaerobic fungi24. The findings of

Tovar et al.7 and Mai et al.8 have re-vealed another organelle that probablyarose from the same source. Futurestudies need to include an increasingnumber of unicellular eukaryotes inorder to uncover the varied endpointsof mitochondrial diversification and re-construct the evolutionary events lead-ing to them25.

AcknowledgementsOriginal research in the laboratory of MM issupported by National Institutes of HealthGrant AI11942 and National ScienceFoundation Grant MBC 9615659.

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and the origin of eukaroytic cells. Mol.Microbiol. 15, 1–11

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19 Simpson, A.G.B. et al. (1997) The organisationof Mastigamoeba schizophrenia n.sp.: moreevidence of ultrastructural idiosyncrasy andsimplicity of pelobiont protists. Eur. J. Protistol.33, 87–98

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21 Embley, T.M. and Hirt, R.P. (1998) Earlybranching eukaryotes? Curr. Opin. Gen. Develop.8, 624–629

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23 Plümper, E. et al. (1998) Implications of proteinimport on the origin of hydrogenosomes.Protist 149, 303–311

24 Hackstein, J.H.P. et al. (1999) Hydrogenosomes:eukaryotic adaptations to anaerobicenvironments. Trends Microbiol. 7, 441–447

25 Embley, T.M. and Martin, W. (1998) Ahydrogen-producing mitochondrion. Nature396, 517–518

Miklós Müller is at the Rockfeller University,1230 York Avenue, New York, NY 10021, USA. Tel: +1 212 327 8153, Fax: +1 212327 7974, e-mail: mmuller@rockvax.rockefeller.edu

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