Eukaryotic evolution: early origin of canonical introns

Spliceosomal introns, one of the hallmarks of eukaryotic genomes, were thought to have originated late in evolution and were assumed not to exist in eukaryotes that diverged early -- until the discovery of a single intron with an aberrant splice boundary in the primitive 'protozoan' Giardia. Here we describe introns from a close relative of Giardia, Carpediemonas membranifera, that have boundary sequences of the normal eukaryotic type, indicating that canonical introns are likely to have arisen very early in eukaryotic evolution.     

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.     

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.     

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.     

The genome of the protist parasite Entamoeba histolytica

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.     

Structural basis of highly conserved ribosome recycling in eukaryotes and archaea

Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.     

Humanin peptide suppresses apoptosis by interfering with Bax activation

Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.     

Quantitative reactivity profiling predicts functional cysteines in proteomes

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.     

Origin of the eukaryotic nucleus determined by rate-invariant analysis of rRNA sequences

The origin of the eukaryotic nucleus is difficult to reconstruct. Eukaryotic organelles (chloroplast, mitochondrion) are eubacterial endosymbionts, but the source of nuclear genes has been obscured by multiple nucleotide substitutions. Using evolutionary parsimony, a newly developed rate-invariant treeing algorithm, the eukaryotic ribosomal rRNA genes are shown to have evolved from the eocytes, a group of extremely thermophilic, sulphur-metabolizing, anucleate cells. The deepest bifurcation yet found separates the reconstructed tree into two taxonomic divisions. These are a proto-eukaryotic group (karyotes) and an essentially bacterial one (parkaryotes). Within the precision of the rooting procedure, the tree is not consistent with either the prokaryotic-eukaryotic or the archaebacterial-eubacterial-eukaryotic groupings. It implies that the last common ancestor of extant life, and the early ancestors of eukaryotes, probably lacked nuclei, metabolized sulphur and lived at near-boiling temperatures.     

Characterization and expression of a cDNA, AmphiSDHD , encoding the amphioxus cytochrome b small subunit in mitochondrial succinate ubiquinone oxidoreductase

Succinate ubiquinone oxidoreductase ,alsoknownascomplexII ,isthesmallestenzymecomplexfunctioninginboththetricarboxylicacidcycle(TAC)andtheaerobicrespiratorychainofeukaryoticcellmitochondriaandprokaryoticcells[1] .Itiscom posedoffournuclear encoded proteins ,SDHAorflavoprotein (70kD) ,SDHBoriron sulfurprotein(2 7kD) ,SDHCorCII 3(15kD)andSDHDorCII 4 (7— 9kD)inmosteukaryotes[2 ] .Thetwoproteins ,SDHAandSDHB ,comprisethesuccinatedehydroge nase (SDH) ,anenzymeoftheTACandaperipher…     

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.     

Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1

There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.     

Independent evolution of structural and coding regions in a Neurospora mitochondrial intron

The discovery of intervening sequences (introns) in eukaryotic genes has raised questions about the origin and evolution of these sequences. Hypotheses concerning these topics usually consider the intron as a unit that could be lost or gained over time, or as a region within which recombination can occur to facilitate the production of new proteins by exon shuffling. Additional complexities are observed in introns of mitochondrial and chloroplast genes which contain secondary structures required for messenger RNA splicing and open-reading frames encoding proteins. Here we describe differences in the organization of protein-coding sequences in the intron of the mitochondrial ND1 gene in two closely related species of Neurospora. These differences show that intron sequences involved in secondary structure formation and in protein coding can evolve as physically distinct elements. Indeed, the secondary structure elements of the ND1 intron can contain two different coding sequences located at two different positions within the intron.     

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.