Localization and functionality of microsporidian iron-sulphur cluster assembly proteins

Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes (including humans) that show extreme reduction at the molecular, cellular and biochemical level. Although microsporidia have long been considered as early branching eukaryotes that lack mitochondria, they have recently been shown to contain a tiny mitochondrial remnant called a mitosome. The function of the mitosome is unknown, because microsporidians lack the genes for canonical mitochondrial functions, such as aerobic respiration and haem biosynthesis. However, microsporidial genomes encode several components of the mitochondrial iron-sulphur (Fe-S) cluster assembly machinery. Here we provide experimental insights into the metabolic function and localization of these proteins. We cloned, functionally characterized and localized homologues of several central mitochondrial Fe-S cluster assembly components for the microsporidians Encephalitozoon cuniculi and Trachipleistophora hominis. Several microsporidial proteins can functionally replace their yeast counterparts in Fe-S protein biogenesis. In E. cuniculi, the iron (frataxin) and sulphur (cysteine desulphurase, Nfs1) donors and the scaffold protein (Isu1) co-localize with mitochondrial Hsp70 to the mitosome, consistent with it being the functional site for Fe-S cluster biosynthesis. In T. hominis, mitochondrial Hsp70 and the essential sulphur donor (Nfs1) are still in the mitosome, but surprisingly the main pools of Isu1 and frataxin are cytosolic, creating a conundrum of how these key components of Fe-S cluster biosynthesis coordinate their function. Together, our studies identify the essential biosynthetic process of Fe-S protein assembly as a key function of microsporidian mitosomes.     

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.     

An anaerobic mitochondrion that produces hydrogen

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.     

Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex

Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.     

Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation

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.     

Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I

Hydrogenosomes are double-membraned ATP-producing and hydrogen-producing organelles of diverse anaerobic eukaryotes. In some versions of endosymbiotic theory they are suggested to be homologues of mitochondria, but alternative views suggest they arose from an anaerobic bacterium that was distinct from the mitochondrial endosymbiont. Here we show that the 51-kDa and 24-kDa subunits of the NADH dehydrogenase module in complex I, the first step in the mitochondrial respiratory chain, are active in hydrogenosomes of Trichomonas vaginalis. Like mitochondrial NADH dehydrogenase, the purified Trichomonas enzyme can reduce a variety of electron carriers including ubiquinone, but unlike the mitochondrial enzyme it can also reduce ferredoxin, the electron carrier used for hydrogen production. The presence of NADH dehydrogenase solves the long-standing conundrum of how hydrogenosomes regenerate NAD+ after malate oxidation. Phylogenetic analyses show that the Trichomonas 51-kDa homologue shares common ancestry with the mitochondrial enzyme. Recruitment of complex I subunits into a H2-producing pathway provides evidence that mitochondria and hydrogenosomes are aerobic and anaerobic homologues of the same endosymbiotically derived organelle.     

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.     

线粒体蛋白组研究进展

线粒体蛋白组是指在线粒体中出现的所有蛋白质的集合,包括线粒体自身基因组编码的和 由核基因组编码的蛋白质.线粒体作为真核生物的重要细胞器,它参与去除氧化、产生能量和还原 性物质等等一些重要的生命活动过程.这些功能都依赖于线粒体蛋白组中的蛋白河的相互作用.要 对其有一个较为全面的认识,就必须对其蛋白组进行广泛且深入的研究.根据现有的一些研究成 果,对线粒体蛋白组在起源与进化、跨膜运输、研究方法和计算机预测作了简要综述.     

Evolutionary status of<Emphasis Type="Italic">Entamoeba</Emphasis>

In addition to its medical importance as parasitic pathogen, Entamoeba has aroused people‘s interest in its evolutionary status for a long time. Lacking mitochondrion and other intracellular organelles common to typical eukaryotes, Entamoeba and several other amitochondrial protozoans have been recognized as ancient pre-mitochondriate eukaryotes and named “archezoa“, the most primitive extant eukaryotes. It was suggested that they might be living fossils that remained in a primitive stage of evolution before acquisition of organelles, lying close to the transition between prokaryotes and eukaryotes. However, recent studies revealed that Entamoeba contained an organelle, “crypton“ or “mitosome“, which was regarded as specialized or reductive mitochondrion. Relative molecular phylogenetic analyses also indicated the existence or the probable existence of mitochondrion in Entamoeba. Our phylogenetic analysis based on DNA topoisomerase Ⅱ strongly suggested its divergence after some mitchondriate enkaryotes. Here, all these recent researches are reviewed and the evolutionary status of Entamoeba is discussed.     

Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes

The microsporidia are a group of unusual, obligately parasitic protists that infect a great variety of other eukaryotes, including vertebrates, arthropods, molluscs, annelids, nematodes, cnidaria and even various ciliates, myxosporidia and gregarines. They possess a number of unusual cytological and molecular characteristics. Their nuclear division is considered to be primitive, they have no mitochondria, their ribosomes and ribosomal RNAs are reported to be of prokaryotic size and their large ribosomal subunit contains no 5.8S rRNA. The uniqueness of the microsporidia may reflect their phylogenetic position, because comparative sequence analysis shows that the small subunit rRNA of the microsporidium Vairimorpha necatrix is more unlike those of other eukaryotes than any known eukaryote 18S rRNA sequence. We conclude that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.     

A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast

Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles. In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication. We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein. SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein. The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E. coli DnaJ protein. Hybridization experiments indicate that there is a family of yeast genes related to SCJ1. These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.     

Role of Bax and Bak in mitochondrial morphogenesis

Bcl-2 family proteins are potent regulators of programmed cell death. Although their intracellular localization to mitochondria and the endoplasmic reticulum has focused research on these organelles, how they function remains unknown. Two members of the Bcl-2 family, Bax and Bak, change intracellular location early in the promotion of apoptosis to concentrate in focal clusters at sites of mitochondrial division. Here we report that in healthy cells Bax or Bak is required for normal fusion of mitochondria into elongated tubules. Bax seems to induce mitochondrial fusion by activating assembly of the large GTPase Mfn2 and changing its submitochondrial distribution and membrane mobility-properties that correlate with different GTP-bound states of Mfn2. Our results show that Bax and Bak regulate mitochondrial dynamics in healthy cells and indicate that Bcl-2 family members may also regulate apoptosis through organelle morphogenesis machineries.     

Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I

Eukaryotic cells are dependent on their ability to translocate membraneous elements about the cytoplasm. In many cells long translocations of organelles are associated with microtubules. In other cases, such as the rapid cytoplasmic streaming in some algae, organelles appear to be propelled along actin filaments. It has been assumed, but not proven, that myosin produces these movements. We have tested vesicles from another eukaryotic cell for their ability to move on the exposed actin bundles of Nitella as an indiction that actin-based organelle movements may be a general property of cells. We found that organelles from Acanthamoeba castellanii can move along Nitella actin filaments. Here, we report two different experiments indicating that the single-headed non-polymerizable myosin isozyme myosin-I is responsible for this organelle motility. First, monoclonal antibodies to myosin-I inhibit movement, but antibodies that inhibit double-headed myosin-II do not. Second, approximately 20% of the myosin-I in homogenates co-migrates with motile vesicles during Percoll density-gradient ultracentrifugation. This is the first indication of a role for myosin-I within the cell and supports the suggestion of Albanesi et al. that myosin-I moves vesicles in this way.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

Mitochondrial function in normal and diabetic beta-cells.

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic beta-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on beta-cell function reflects the importance of mitochondria in the control of insulin secretion. The beta-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger.     

Independent transfer of mitochondrial chromosomes and plasmids during unstable vegetative fusion in Neurospora

The sporadic distribution of similar introns in organelle, nuclear ribosomal RNA and bacteriophage genes suggests that at least some of these introns are mobile genetic elements. Some plasmids in fungal mitochondria contain intron-like sequences and, like introns, they have scattered distributions within and among species. The occurrence and evolutionary importance of such horizontal transfer of DNA, not only between fungi, but among a wide range of organisms have been matters of much discussion. Here, we report experimental evidence for transfer of Neurospora mitochondrial plasmids from one mitochondrial genotype to another at high frequency during unstable vegetative cell fusion. Exchange of mitochondrial and nuclear genomes can also occur. These observations suggest that vegetative fusion may have a more important role in the mitochondrial genetic structure of natural populations than is generally thought, and may provide an explanation for the distribution of certain plasmids and possibly other mobile genetic elements.     

The mitochondrial chaperonin hsp60 is required for its own assembly

Heatshock protein 60 (hsp60) in the matrix of mitochondria is essential for the folding and assembly of newly imported proteins. Hsp60 belongs to a class of structurally related chaperonins found in organelles of endosymbiotic origin and in the bacterial cytosol. Hsp60 monomers form a complex arranged as two stacked 7-mer rings. This 14-mer complex binds unfolded proteins at its surface, then seems to catalyse their folding in an ATP-dependent process. The question arises as to how such an assembly machinery is itself folded and assembled. Hsp60 subunits are encoded by a nuclear gene and translated in the cytosol as precursors which are translocated into mitochondria and proteolytically processed. In both intact cells and isolated mitochondria of the hsp60-defective yeast mutant mif4, self-assembly of newly imported wild-type subunits is not observed. Functional pre-existing hsp60 complex is required in order to form new, assembled, 14-mer. Subunits imported in vitro are assembled with a surprisingly fast half-time of 5-10 min, indicative of a catalysed reaction. These findings are further evidence that self-assembly may not be the principal mechanism by which proteins attain their functional conformation in the intact cell.     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.     

Complete replacement of mitochondrial DNA in Drosophila

The introduction of foreign mitochondria or mitochondrial DNA into a cell is a useful technique for clarifying the molecular mechanisms responsible for the maintenance of mitochondria. Novel combinations of mitochondrial and nuclear genomes have been studied in mammalian cells in culture and in yeast. In Drosophila, we have recently constructed heteroplasmic flies possessing both endogenous mitochondrial DNA and foreign mitochondrial DNA by intra- and interspecific transplantation of germ plasm. During the maintenance of these heteroplasmic lines, flies of D. melanogaster are produced that no longer possess their own mitochondrial DNA but retain the foreign mitochondrial DNA from D. mauritiana. .These flies are fertile and the foreign mitochondrial DNA is stably maintained in their offspring. Here we report the complete replacement of endogenous mitochondrial DNA with that from another multicellular species. Molecular and genetic analysis of this replacement in Drosophila should provide new insight into the functional interaction between nuclear and organelle genomes.