A mitochondrial remnant in the microsporidian Trachipleistophora hominis

Microsporidia are obligate intracellular parasites of several eukaryotes. They have a highly complex and unique infection apparatus but otherwise appear structurally simple. Microsporidia are thought to lack typical eukaryotic organelles, such as mitochondria and peroxisomes. This has been interpreted as support for the hypothesis that these peculiar eukaryotes diverged before the mitochondrial endosymbiosis, which would make them one of the earliest offshoots in eukaryotic evolution. But microsporidial nuclear genes that encode orthologues of typical mitochondrial heatshock Hsp70 proteins have been detected, which provides evidence for secondary loss of the organelle or endosymbiont. In addition, gene trees and more sophisticated phylogenetic analyses have recovered microsporidia as the relatives of fungi, rather than as basal eukaryotes. Here we show that a highly specific antibody raised against a Trachipleistophora hominis Hsp70 protein detects the presence, under light and electron microscopy, of numerous tiny ( approximately 50 x 90 nm) organelles with double membranes in this human microsporidial parasite. The finding of relictual mitochondria in microsporidia provides further evidence of the reluctance of eukaryotes to lose the mitochondrial organelle, even when its canonical function of aerobic respiration has been apparently lost.     

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.     

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.     

Mitoferrin is essential for erythroid iron assimilation

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.     

Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis

Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly.     

Identification of a mammalian mitochondrial porphyrin transporter

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.     

Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2)?→?2H(+)?+?2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32??. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.     

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.     

Hsp90 stress potentiates rapid cellular adaptation through induction of aneuploidy

Aneuploidy--the state of having uneven numbers of chromosomes--is a hallmark of cancer and a feature identified in yeast from diverse habitats. Recent studies have shown that aneuploidy is a form of large-effect mutation that is able to confer adaptive phenotypes under diverse stress conditions. Here we investigate whether pleiotropic stress could induce aneuploidy in budding yeast (Saccharomyces cerevisae). We show that whereas diverse stress conditions can induce an increase in chromosome instability, proteotoxic stress, caused by transient Hsp90 (also known as Hsp82 or Hsc82) inhibition or heat shock, markedly increased chromosome instability to produce a cell population with high karyotype diversity. The induced chromosome instability is linked to an evolutionarily conserved role for the Hsp90 chaperone complex in kinetochore assembly. Continued growth in the presence of an Hsp90 inhibitor resulted in the emergence of drug-resistant colonies with chromosome XV gain. This drug-resistance phenotype is a quantitative trait involving copy number increases of at least two genes located on chromosome XV. Short-term exposure to Hsp90 stress potentiated fast adaptation to unrelated cytotoxic compounds by means of different aneuploid chromosome stoichiometries. These findings demonstrate that aneuploidy is a form of stress-inducible mutation in eukaryotes, capable of fuelling rapid phenotypic evolution and drug resistance, and reveal a new role for Hsp90 in regulating the emergence of adaptive traits under stress.     

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.     

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.     

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.     

Evolutionary capacitance as a general feature of complex gene networks

An evolutionary capacitor buffers genotypic variation under normal conditions, thereby promoting the accumulation of hidden polymorphism. But it occasionally fails, thereby revealing this variation phenotypically. The principal example of an evolutionary capacitor is Hsp90, a molecular chaperone that targets an important set of signal transduction proteins. Experiments in Drosophila and Arabidopsis have demonstrated three key properties of Hsp90: (1) it suppresses phenotypic variation under normal conditions and releases this variation when functionally compromised; (2) its function is overwhelmed by environmental stress; and (3) it exerts pleiotropic effects on key developmental processes. But whether these properties necessarily make Hsp90 a significant and unique facilitator of adaptation is unclear. Here we use numerical simulations of complex gene networks, as well as genome-scale expression data from yeast single-gene deletion strains, to present a mechanism that extends the scope of evolutionary capacitance beyond the action of Hsp90 alone. We illustrate that most, and perhaps all, genes reveal phenotypic variation when functionally compromised, and that the availability of loss-of-function mutations accelerates adaptation to a new optimum phenotype. However, this effect does not require the mutations to be conditional on the environment. Thus, there might exist a large class of evolutionary capacitors whose effects on phenotypic variation complement the systemic, environment-induced effects of Hsp90.     

Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria

Only five mitochondrial proteins are known to be essential for viability of the yeast Saccharomyces cerevisiae; all of them are key components of the mitochondrial protein import system. Other components of this system are not essential for life; they include functionally redundant import receptors on the mitochondrial surface and enzymes acting upon only a few precursor proteins.     

近红外光谱-系统聚类法快速测定煤炭品质

采用偏最小二乘法对95个煤炭样品的近红外光谱数据进行处理,并提取主成分.将提取的主成分与煤炭的发热量、灰分、挥发份、含硫量和全水分共同作为变量,进行系统聚类分析.将样品数据聚类为4组,同时剔除异常样本.对聚类后的各组数据采用多元散射校正、二阶导数、诺里斯导数平滑进行预处理,建立偏最小二乘定量分析模型.采用逐步筛选法,求得以发热量为变量的Bayes判别函数,交互验证结果表明判别函数稳定性良好.对未知样品发热量、灰分、挥发份、含硫量和全水分预测的决定系数分别达到0.992、0.927、0.938、0.778、0.978,说明模型预测性能良好.     

微生物线粒体衍生细胞器及产氢体研究进展

对线粒体衍生细胞器的动基体、线性体和隐性体以及产氢体的形态、结构、功能和起源等方面进行了综述,并总结了近十年来线粒体衍生细胞器及产氢体的研究进展.     

Synexpression groups in eukaryotes

In 1960, Jacob and Monod described the bacterial operon, a cluster of functionally interacting genes whose expression is tightly coordinated. Global expression analysis has shown that the highly coordinate expression of genes functioning in common processes is also a widespread phenomenon in eukaryotes. These sets of co-regulated genes, or 'synexpression groups', show a striking parallel to the operon, and may be a key determinant facilitating evolutionary change leading to animal diversity.     

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.