Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year. Giardia is a member of the diplomonads, often described as an ancient protist group whose primitive nature is suggested by the lack of typical eukaryotic organelles (for example, mitochondria, peroxisomes), the presence of a poorly developed endomembrane system and by their early branching in a number of gene phylogenies. The discovery of nuclear genes of putative mitochondrial ancestry in Giardia and the recent identification of mitochondrial remnant organelles in amitochondrial protists such as Entamoeba histolytica and Trachipleistophora hominis suggest that the eukaryotic amitochondrial state is not a primitive condition but is rather the result of reductive evolution. Using an in vitro protein reconstitution assay and specific antibodies against IscS and IscU--two mitochondrial marker proteins involved in iron-sulphur cluster biosynthesis--here we demonstrate that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron-sulphur protein maturation. Our results indicate that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont.

Mitochondrial remnant organelles (mitosomes) that exist in a range of "amitochondrial" eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in ironsulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically.Highly derived but functional mitochondrial remnant organelles (mitosomes) are found in "amitochondrial" eukaryotes as diverse as Entamoeba histolytica, Trachipleistophora hominis, Cryptosporidium parvum, Blastocystis hominis, and Giardia intestinalis (1-5). Although able to metabolize small amounts of oxygen, these organisms live mostly in oxygendeprived environments and have lost their capacity for ATP biosynthesis linked to oxidative phosphorylation, one of the main functions of aerobic mitochondria (6). Investigating the biology of these vestigial organelles is thus central to our understanding of mitochondrial evolution and of the minimal set of proteins required for the biogenesis and inheritance of an endosymbiosis-derived organelle (7-9).Giardia lamblia is a unicellular protozoan parasite of the small intestine in vertebrates and a leading cause of diarrheal disease worldwide (10). It may also represent the most basal branch of eukaryotic evolution, although its phylogenetic placing remains controversial (11-13). The presence of a poorly developed endomembrane system and its unusual genomic organization make Giardia an ideal model for the study of early genome organization and cell biology (14, 15). Indirect evidence that Giardia once harbored a mitochondrion (16) appeared to be confirmed by the discovery of small (ϳ100 nm) double membrane-bounded organelles distributed throughout the cytoplasm that harbor enzymes that participate in iron-sulfur cluster assembly, an essential function of mitochondria (3, 17). However, attempts to localize the mitochondrial marker Cpn60 using heterologous anti-Cpn60 antibodies proved controversial, as the observed label did not seem to be associated with biological membranes...

Recent data suggest that microaerophilic and parasitic protozoa, which lack oxidative phosphorylation, nevertheless contain mitochondrial homologs [1-6], organelles that share common ancestry with mitochondria. Such widespread retention suggests there may be a common function for mitochondrial homologs that makes them essential for eukaryotic cells. We determined the mitochondrial carrier family (MCF) complement of the Entamoeba histolytica mitochondrial homolog, also known as a crypton [5] or more commonly as a mitosome [3]. MCF proteins support mitochondrial metabolic energy generation, DNA replication, and amino-acid metabolism by linking biochemical pathways in the mitochondrial matrix with those in the cytosol [7]. MCF diversity thus closely mirrors important facets of mitochondrial metabolic diversity. The Entamoeba histolytica mitosome has lost all but a single type of MCF protein, which transports ATP and ADP via a novel mechanism that is not reliant on a membrane potential. Phylogenetic analyses confirm that the Entamoeba ADP/ATP carrier is distinct from archetypal mitochondrial ADP/ATP carriers, an observation that is supported by its different substrate and inhibitor specificity. Because many functions of yeast and human mitochondria rely on solutes transported by specialized members of this family, the Entamoeba mitosome must contain only a small subset of these processes requiring adenine nucleotide exchange.

The existence of mitochondrion-related relict organelles (mitosomes) in the amitochondrial human pathogen Entamoeba histolytica and the detection of extranuclear DNA-containing cytoplasmic structures (EhKOs) has led to the suggestion that a remnant genome from the original mitochondrial endosymbiont might have been retained in this organism. This study reports on the mutually exclusive distribution of Cpn60 and extranuclear DNA in E. histolytica and on the distribution of Cpn60-containing mitosomes in this parasite. In situ nick-translation coupled to immunofluorescence microscopy failed to detect the presence of DNA in mitosomes, either in fixed parasite trophozoites or in partially purified organellar fractions. These results indicate that a remnant organellar genome has not been retained in E. histolytica mitosomes and demonstrate unequivocally that EhKOs and mitosomes are distinct and unrelated cellular structures. INTRODUCTIONOver the past few years mitochondrial remnant organelles (mitosomes) have been identified in a number of amitochondrial parasitic protozoa (e.g. Giardia intestinalis, Entamoeba histolytica, Trachipleistophora hominis and Cryptosporidium parvum) (Mai et al., 1999;Tovar et al., 1999Tovar et al., , 2003Williams et al., 2002;Riordan et al., 2003), disproving the Archezoa hypothesis, which postulated that amitochondrial eukaryotic organisms were the direct descendants of the nucleated cell that hosted the original mitochondrial endosymbiont (Cavalier-Smith, 1983, 1998. It is now apparent that eukaryotic microbes that lack typical mitochondria are not primitively amitochondrial but are highly derived descendants of mitochondrioncontaining ancestors whose capacity for aerobic respiration has been gradually lost during the course of evolution.Because of their recent discovery, little is known about the physiological functions of mitosomes. Some, such as those of Entamoeba and Cryptosporidium, have been shown to contain chaperonin Cpn60, a protein known to participate in the refolding of imported proteins (Sigler et al., 1998; Mai et al., 1999;Tovar et al., 1999;Riordan et al., 2003); mitosomes of Trachipleistophora contain a mitochondrialtype Hsp70 (mtHsp70), a molecular motor that helps internalize proteins into the organelle (Matouschek et al., 2000;Williams et al., 2002). Other proteins have also been suggested as putative mitosomal components in various amitochondrial lineages but their cellular localization has not been demonstrated experimentally (Clark & Roger, 1995;Bakatselou et al., 2000Bakatselou et al., , 2003Katinka et al., 2001;Morrison et al., 2001;Zhu & Keithly, 2002;Arisue et al., 2002). Perhaps the most significant finding in relation to the biology of mitosomes is the direct demonstration that Giardia mitosomes function in the biosynthesis of molecular iron-sulphur (Fe-S) clusters and in their subsequent incorporation into functional Fe-S proteins (Tovar et al., 2003). Genes encoding several proteins involved in Fe-S cluster metabolism have also been identified in the genome...

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