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Pregunta 3: la razón es que los anticuerpos fluorescentes son invisibles en el microscopio electrónico, y en cambio las partículas de oro impiden el paso de los electrones y por lo tanto se pueden visualizar

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Pregunta 3: la razón es que los anticuerpos fluorescentes son invisibles en el microscopio electrónico, y en cambio las partículas de oro impiden el paso de los electrones y por lo tanto se pueden visualizar

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Aislamiento de mitocondrias intactas del músculo esquelético mediante centrifugación diferencial para mediciones de respirometría de alta resolución

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Aislamiento de mitocondrias intactas del músculo esquelético mediante centrifugación diferencial para mediciones de respirometría de alta resolución

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Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements - PubMed

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Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by…

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Integrantes : Laura Rodríguez, Santiago Cabrera, Angélica Flores, Miguel Flores,  Vanessa Falcón y Alejandro Triviño

¿Por qué fue importante el paso de centrifugación a baja velocidad?

La centrifugación a baja velocidad alcanza una velocidad máxima de 5000 rpm, se la puede utilizar sin o con refrigeración y se utiliza para la separación de grandes partículas la cual es sedimentada. Y las partículas más pequeñas requieren velocidades mayores y periodos más largos.

Sugiera por qué sería necesario preservar químicamente las células antes de diseccionarlos.

Para poder preservar la célula se necesita conservar o proteger la degradación del ácido ribonucleico contenido en células antes de separar el ARN, su conservación normalmente se utiliza para análisis cuantitativo o cualitativo, ensayos de protección de la nucleasa y RT-PCR y es fundamental para realizar ensayos de biología molecular y experimentos como hibridación Norther.

¿Por qué fue necesario usar anticuerpos marcados con oro en lugar de anticuerpos marcados con fluorescencia en este trabajo?

Porque en la técnica inmunológica de inmunocromatografía se visualiza la reacción antígeno-anticuerpo mediante la acumulación del oro coloidal del conjugado en zonas específicas del papel de nitrocelulosa donde se fijan previamente anticuerpos de captura. El complejo de oro coloidal con los antígenos migran sobre el papel de nitrocelulosa para reaccionar específicamente con los anticuerpos inmovilizados en el papel, dando bandas coloreadas por la acumulación del oro coloidal del conjugado. El papel de nitrocelulosa permite la migración por capilaridad de líquidos y solutos y a la vez conserva activos los anticuerpos fijados.

¿Cuál es el tamaño aproximado (en kDa) de la proteína IscS?

Según la base de datos Unit Pro 44.6 kDa.

Los medicamentos que se usan para tratar las infecciones por Giardia funcionan al ingresar al citoplasma de trofozoíto e interrumpiendo la producción de ATP. ¿Por qué tal droga puede ser seguro para su uso en humanos y animales?

Segun lo consultado,no ha existido muertes por usar antiparasitario,a diferencia de que una persona o animal no se someta a un tratamiento antidesparasitario,lo cual si le puede causar una amebiasis cronica lo que puede conllevar a la muertes.De igual manera el farmaco menos invasivo para tratar la afeccion por Giardia es el albedanzol o metronidazol,siendo estos los farmacos de primera linea en donde actua inhibiendo la sintesis de ATP en las celulas de este protozoo,pero como todo farmaco va a tener sus efectos secundarios,los cuales no son tan fuertes.Todo dependera del metabolismo de la persona,de como pueda ser asimilado por su organismo,aunque los antidesparasitario siempre se van a sintetizar en el higado,lo que de igual manera se recomienda no ingerir alcohol cuando se esta consumiendo antidesparasitario,ya que el etanol acelera su sintesis produciendo efectos secundarios mas fuertes para la salud.

Investigar/revisar el papel de la estructura de la proteína en la función de un anticuerpo.

Las proteínas son las que determinan la forma y estructura de las células,  dirigen los procesos vitales y son específicas. Según la estructura de las mismas pueden unirse bien sea a otra proteína o incluso a otras moléculas distintas como es el caso de los anticuerpos a los antígenos, a estas se las conoce como inmunoglobulinas.

Investigar/revisar la evidencia que apoya el origen endosimbiótico de mitocondrias

La teoría endosimbiótica o de endosimbiosis seriada fue presentada por Lynn Margulis en 1967. Además de las mitocondrias, se aplica también a los cloroplastos, explica por qué mitocondrias y cloroplastos tienen ADN original de la bacteria, entre los hechos que apoyan a esta teoría podemos mencionar que las mitocondrias son  muy similares a bacterias actuales, tienen tamaño parecido, contienen ADN circular bicatenario cerrado, tienen ribosomas 70S (a diferencia de los 80S de las células eucariotas que las rodean), también se dividen por fusion binaria, además de tener una doble membrana , lo cual es signo que la mitocondria fue fagocitada, por otra parte se han encontrado similitudes entre el ADN mitocondrial y nuclear, cabe mencionar que las mitocondrias son incapaces de sobrevivir fuera de la célula eucariota.

¿En qué parte de la célula del trofozoíto se encuentran las proteínas Isc?

La distribución de proteínas Isc se puede observar gracias a la microscopía de fluorescencia de barrido confocal, se observó que estaban localizadas en pequeñas estructuras celulares distribuidas por todo el citoplasma, incluyendo alrededor de los cuerpos basales y la base de los axonemas flagelares.

Los medicamentos que se usan para tratar las infecciones por Giardia funcionan al ingresar al citoplasma de trofozoíto e interrumpiendo la producción de ATP. ¿Por qué tal droga puede ser seguro para su uso en humanos y animales?

Según lo consultado,no ha existido muertes por usar antiparasitario,a diferencia de que una persona o animal no se someta a un tratamiento anti desparasitario,lo cual si le puede causar una amebiasis crónica lo que puede conllevar a la muertes.De igual manera el fármaco menos invasivo para tratar la afeccion por Giardia es el albendazol o metronidazol,siendo estos los fármacos de primera línea en donde actúa inhibiendo la síntesis de ATP en las células de este protozoo,pero como todo fármaco va a tener sus efectos secundarios,los cuales no son tan fuertes.Todo depende del metabolismo de la persona,de como pueda ser asimilado por su organismo,aunque los anti desparasitario siempre se van a sintetizar en el hígado,lo que de igual manera se recomienda no ingerir alcohol cuando se esta consumiendo anti desparasitario,ya que el etanol acelera su síntesis produciendo efectos secundarios más fuertes para la salud.

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researchgate.net/profile/Marcelo-Arana-2/publication/261252367_Clasificacion_y_Nomenclatura_de_los_seres_vivos/links/596a189aa6fdcc18ea74ee76/Clasificacion-y-Nomenclatura-de-los-seres-vivos.pdf

Bianco, C., Basconsuelo, S., & Malpassi, R. (2011). El misterio de la vida. Compilado de Biología para ingresantes a la universidad. UniRio, Río Cuarto.

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Gallardo, M. E., & Garesse, R. (2012). Origen y evolución de la mitocondria: ADN mitocondrial y evolución humana. Monografías de la Real Academia Nacional de Fa

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Origen y evolución de la mitocondria: ADN mitocondrial y evolución humana

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Origen y evolución de la mitocondria: ADN mitocondrial y evolución humana

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Mitochondrial diseases: An applied way of teaching genetics.

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Mitochondrial diseases: An applied way of teaching genetics.

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Abstract:

[ES]Este trabajo trata las enfermedades mitocondriales como "vehículo" para enseñar genética, biotecnología y salud. Se pretende facilitar la asimilación de estos conceptos, cambiando los tópicos que suelen aparecer en los institutos, por una genética más simple y actualizada. El tema a tratar se inserta en una Unidad Didáctica dentro de la asignatura "ciencias para el mundo contemporáneo", de 1º de Bachillerato. Se propone que los alumnos vean la genética de una forma práctica, innovadora, aplicada y atractiva a través de temas de actualidad, las enfermedades mitocondriales. Al poseer un genoma mucho más simple, un alumno puede estudiar la genética por comparación entre el funcionamiento de la mitocondria y el funcionamiento del núcleo celular, mejorando así su comprensión de todos estos procesos. Además, el tema nos permite desarrollar conceptos relacionados con a salud, por lo que conseguimos una interrelación entre unidades de temario.
[EN]This paper deals with mitochondrial diseases as a "vehicle" to teach genetics, biotechnology and health. It is intended to facilitate the assimilation of these concepts by changing the topics that usually appear in high schools, for a simpler and updtated genetics. The theme will be inserted into a teaching unit within the subject "Science for the contemporary world" of 1st year of High School educational stage. It is proposed that the students see the genetics in a practical, innovative, attractive and applied way through topics, mitochondrial diseases. Possesing a much simpler genome, a student can study genetic comparing the operation of the mitochondria and the operation of the nucleus, thus enhacing their understanding of all theses processes. In addition, the issue allows us to develop concepts related to health, so we got a relation ship between units.

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Trabajos Academicos de la Universidad de Jaen: Enfermedades mitocondriales: Una forma aplicada de enseñar genética

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ES]Este trabajo trata las enfermedades mitocondriales como "vehículo" para enseñar genética, biotecnología y salud. Se pretende facilitar la asimilación de estos conceptos, cambiando los tópicos que suelen aparecer en los institutos, por una genética más simple y actualizada. El tema a tratar se inserta en una Unidad Didáctica dentro de la asignatura "ciencias para el mundo contemporáneo", de 1º de Bachillerato.

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Indagando en el origen de las mitocondrias

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Indagando en el origen de las mitocondrias

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Riera, A. M. Indagando en el origen de las mitocondrias. https://revistes.ub.edu/index.php/b_on/article/download/10096/12969/0

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Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

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Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

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https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1000243

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Ondřej Šmíd,

Anna Matušková,

Simon R. Harris,

Tomáš Kučera,

Marián Novotný,

Lenka Horváthová,

Ivan Hrdý,

Eva Kutějová,

Robert P. Hirt,

T. Martin Embley

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Abstract

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.

Author Summary

In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called α or β, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis. The Giardia enzyme is unique among eukaryotes because it has undergone reductive evolution to function efficiently as a single β-subunit monomer. We also show that the recent claim that the equivalent enzyme in the hydrogenosome, another type of reduced mitochondrion of the human parasite Trichomonas vaginalis, functions as a homodimer of two β-subunits, is not supported. The Trichomonas enzyme requires both an α- and a β-subunit to function most efficiently. Computational analysis of the Giardia and Trichomonas enzymes reveals that their structures and surface charge distributions have co-evolved to match the peculiar properties of the targeting signals that they process. The Giardia mitosome is an ideal model for studying the limits of mitochondrial reductive evolution and, because it makes cofactors that are essential for Giardia survival, is a potential therapeutic target for this important human parasite.

Citation: Šmíd O, Matušková A, Harris SR, Kučera T, Novotný M, Horváthová L, et al. (2008) Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis. PLoS Pathog 4(12): e1000243. https://doi.org/10.1371/journal.ppat.1000243

Editor: Daniel Eliot Goldberg, Washington University School of Medicine, United States of America

Received: September 8, 2008; Accepted: November 18, 2008; Published: December 19, 2008

Copyright: © 2008 Šmíd et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The project was funded by the Grant Agency of the Academy of Sciences of the CR IIA501110631 (J.T.), Ministry of Education, Youth and Sports of the CR MSM0021620858 and LC07032 (J.T.), and the Grant Agency of Charles University B-Bio166/2006 (O.S.). S.H., R.P.H., and T.M.E. were supported by a grant (BB/C006143/1) from the Biotechnology and Biological Sciences Research Council (UK) and funding from the Royal Society (UK) to T.M.E.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The acquisition of the mitochondrial endosymbiont and its evolution into the mitochondrion were key events in the evolution of eukaryotes [1]. During this process, most of the protomitochondrial genome was either lost or transferred to the nucleus of the host cell [2]. As a consequence, most mitochondrial proteins are host-nuclear encoded and must be specifically targeted to the organelle where they function. In the best understood system, N-terminal extensions attached to mitochondrial matrix proteins are specifically recognised by receptors on the mitochondrial surface, and the preproteins are subsequently imported by translocases of the outer and inner mitochondrial membranes [3]. A final step in the import process is the removal of the N-terminal extension, by the mitochondrial processing peptidase (MPP) [4], to prevent it from interfering with protein function and/or stability [5]. The MPP comprises a catalytic βMPP subunit that binds a zinc cation using amino acid residues of the conserved motif H XXEH X ₇₆E [6], and a regulatory αMPP subunit with a flexible glycine-rich loop that is important for substrate recognition [7]. The two subunits together form a negatively charged cavity that accommodates and immobilizes presequences during processing [6]. The activity of MPP thus requires the cooperative action of both subunits; neither subunit is functional alone [6],[8].

Mitochondrial targeting presequences are characterized by the ability to form a positively charged amphipathic alpha helix, but otherwise show little primary sequence conservation [6]. Their most prominent common feature is the presence of a cleavage motif, which determines the peptide bond to be cleaved by the processing peptidase. The cleavage motif includes a positively charged residue, typically arginine, at the -2 or -3 position from the cleavage site (P ₂ or P ₃), which is followed by hydrophobic (P ₁′) and hydrophilic (P ₂′, P ₃′) residues [9]. Mutational analyses indicate that the P ₂ (P ₃) arginine plays a key role in the recognition of the processing site by MPP and interacts with the glutamate of the βMPP active site [9]. In addition, there are one or more basic amino acid residue(s) N-terminally distal from the processing site that bind to acidic residues of the MPP cavity and stabilize the substrate-MPP complex [10].

Mitosomes and hydrogenosomes are highly reduced versions of mitochondria that are found in diverse parasitic or free-living unicellular eukaryotes inhabiting oxygen-poor or intracellular niches [1]. The organelles found in human parasites Giardia intestinalis and Trichomonas vaginalis lack a genome so all of their proteins are encoded by the nuclear genome and must be imported [1]. Some hydrogenosomal and mitosomal proteins have N-terminal extensions that are reminiscent of the presequences that direct proteins into mitochondria and they contain distinguishable cleavage motifs [11],[12]. This suggests that the Giardia and Trichomonas organelles may also contain an MPP-like enzyme. A single gene coding for a putative processing peptidase has been found in the genome of G. intestinalis [13] and the gene product has been shown to localize in mitosomes [14]. The primary structure of GPP is highly divergent from mitochondrial homologues, with only 13.1% identity and 29.7% similarity to the βMPP of Saccharomyces cerevisiae. A single gene for a βMPP homologue (20.9% identity and 42.9% similarity to S. cerevisiae βMPP) was also recently identified in the genome of T. vaginalis [15]. In this case, functional data were presented suggesting that the hydrogenosomal processing peptidase (βHPP) functioned as a homodimeric enzyme [15]. No αMPP homologue was detected, although a protein rich in glycine amino acid residues (GRLP), that shares a limited similarity with the glycine-rich loop of αMPP, was located to T. vaginalis hydrogenosomes. However, GRLP was reported not to stimulate βHPP activity in vitro [15].

The progenitor of MPP was probably a monomeric α-proteobacterial peptidase, similar to the recently described Rickettsia prowazekii processing peptidase (RPP) [16]. During the evolution of mitochondria, gene duplication and subunit specialization gave rise to the heterodimeric α/βMPP, which is now present in the mitochondrial matrix or integrated as the core I and II subunits of the cytochrome bc1 complex in the inner mitochondrial membrane [6]. The single subunit structure of GPP and HPP [15] could thus reflect retention of the ancestral form of organization, or reductive evolution from the classic MPP heterodimer. It has also been suggested that the Giardia protein may have had a separate origin by lateral gene transfer from a bacterium other than the mitochondrial endosymbiont [13]. Here we show that GPP functions as a monomer consisting of a single βMPP homologue while HPP, like classical MPP, is fully active only upon heterodimerization of an α and β subunit. Based upon phylogenetic and functional analyses we infer that the unique monomeric structure of the Giardia mitosomal processing peptidase GPP, is the result of reductive, substrate-driven evolution from a heterodimeric progenitor enzyme.

Results/Discussion

Phylogenetic analyses of GPP, βHPP and GRLP

To investigate the origins of the MPP-like proteins of Giardia and Trichomonas and the Trichomonas GRLP we carried out a phylogenetic analysis. As these proteins are heterogeneous for their amino acid compositions, and because a failure to accommodate such heterogeneity can lead to incorrect trees [17], we used a recently described node-discreet-compositional-heterogeneity method to analyze the data [17]. A heterogeneous model comprising 10 composition vectors was found sufficient to produce data of similar composition to the original sequences, as judged by Bayesian posterior predictive simulation [17] ( Fig. S1). Phylogenetic analyses using this model support the hypothesis that GPP, βHPP and βMPP share a common origin. This result contrasts with a previous analysis, using a poorly fitting composition homogeneous model, when GPP was reported to have no phylogenetic affinity with either MPP or the α-proteobacteria [13]. The position of the GPP among βMPP, together with the presence of the catalytic motif H XXEH X ₇₆E, are consistent with the protein being a βMPP-like peptidase (βGPP), and not an αMPP-like protein as currently annotated [13]. Importantly, these data, together with the absence of an αMPP-like protein coding sequence on the Giardia genome, support the hypothesis that the single subunit structure of GPP results from reductive evolution including loss of an αMPP-like subunit. The alternative possibility, that the simple GPP structure reflects retention of the ancestral form of organization, is not supported by our analyses. Our results suggest that αMPP and βMPP probably arose once by a primordial gene duplication at the base of eukaryotes, and that all MPP-like proteins share common ancestry with single subunit enzymes from α-proteobacteria, consistent with an origin from the mitochondrial endosymbiont ( Fig. 1A). Notably, our analyses show that the T. vaginalis GRLP is part of the αMPP clade, suggesting that, contrary to previous claims [15], T. vaginalis may possess a functional homologue (GRLP) of αMPP (henceforth αHPP).

(A) Bayesian phylogenetic analysis of MPP-like protein sequences using a model [17] that allows for across-tree changes in protein amino acid composition. Scale bar indicates estimated substitutions per site. Posterior probabilities of 1.0 are shown as black dots on nodes, and those greater than 0.95 are shown as values. Bacterial MPP homologues are shown in black, αMPP in red and βMPP in blue. Trichomonas α- and βHPPs and Giardia βGPP are highlighted in green. Only α-proteobacterial relationships are shown for bacteria. The fit between the model and the data is shown in Fig. S1 and the full tree with additional details are shown in Fig. S2. (B) Protein size exclusion chromatography of purified recombinant βGPP showing that it elutes as a single peak between 17 and 44 kDa. The activity of βGPP was assayed for cleavage of the targeting presequence of GiiscU for each fraction and the products were separated by SDS-PAGE. Shift in protein mobility indicates cleavage of a presequence. βGPP activity was only detected in fractions from the central peak. (C) Separation of proteins from a mitosome-rich fraction on a sucrose gradient along with molecular size markers. Bands on the immunoblot and SDS-PAGE were quantified by densitometry. The calculated molecular mass of the βGPP monomer is 44.5 kDa. (D) Processing activity of the αHPP-His (lane 1), βHPP-His (lane 2) and corresponding α/βHPP heterodimer (lane 3) with TviscU, showing that the α- and β-subunits are both required for activity. (E) Specific activities were also determined for the βHPP subunit and the α/βHPP heterodimer with a fluorescent substrate based on the T. vaginalis adenylate kinase presequence (n = 3, mean values with s.d.) The activity of the βHPP subunit by itself is at the limit of detection for this assay.

GPP functions as a β monomer while HPP forms an α/β heterodimer

To investigate the functionality of the βGPP, βHPP and αHPP-like proteins, we expressed them in E. coli. The recombinant βGPP processed the N-terminal extensions of Giardia mitosomal ferredoxin (Gifdx) and the iron-sulphur cluster scaffold proteins (GiiscU and GiiscA). The processing activity was demonstrated as a shift in the substrate gel mobility and the cleavage sites were identified by N-terminal amino acid sequencing of the cleaved products ( Fig. 2). The activity of the recombinant βGPP was inhibited by the chelator EDTA, and activity was also lost when the first glutamate of the H XXEH X ₇₆E motif was mutated to glutamine ( Fig. 3). These data indicate that the βGPP is an active metallopeptidase with a similar cleavage mechanism to MPP [6]. Like the rickettsial homologue of MPP [16], βGPP was active as a monomer, which was demonstrated by size exclusion chromatography of recombinant βGPP as well as by analysis of βGPP from a mitosome-rich fraction separated on a sucrose gradient under native conditions ( Fig. 1B, C). Importantly, kinetic parameters of monomeric βGPP (V max = 0.27 µM/min; K m = 8.4 µM, Fig. S3) were comparable to those published for the heterodimeric MPP of Neurospora crassa [8]. It has recently been suggested that the T. vaginalis HPP functions as a homodimer of two identical βHPP subunits [15], so we investigated the activity of βHPP with- and without αHPP. Unlike for βGPP, no activity for βHPP alone could be detected by gel shift assay ( Fig. 1D), but a small amount of activity was observed when a highly sensitive fluorometric assay was used [15] ( Fig. 1E). However, the processing activity measured by this assay increased by almost two orders of magnitude when the βHPP was associated with the αHPP-like protein, indicating that-like classic MPP-the T. vaginalis HPP functions most efficiently as a heterodimer ( Fig. 1E).

The sequence of the demonstrated N-terminal mitochondrial, mitosomal or hydrogenosomal targeting presequences is indicated for each substrate protein with / indicating the cleavage site. Processing of Giardia intestinalis mitosomal presequences (Gifdx, [2Fe2S] ferredoxin; GiiscA and GiiscU, metallochaperones involved in FeS cluster assembly), Trichomonas vaginalis hydrogenosomal presequences (Tvfdx, [2Fe2S] ferredoxin; TvAK, adenylate kinase; Tvhsp70, heat shock protein 70; TviscU, metallochaperone involved in FeS cluster assembly) and mitochondrial presequences (ScMDH, Saccharomyces cerevisiae malate dehydrogenase; MmMDH, Mus musculus MDH; ClMDH, Citrullus lanatus MDH) was tested. Reaction products were separated by SDS-PAGE. Shift in protein mobility indicates cleavage of a targeting presequence. The sites of cleavage indicated by slashes in left column were determined by N-terminal amino acid sequencing. Substrates were incubated with (+) or without (−) the corresponding protease.

(A) Alignment of βGPP and βMPP subunit showing the conserved zinc-binding motif. (B) Effect of protease inhibitors and mutation of E37 on the activity of βGPP. Lane 1: βGPP+GiiscU showing cleavage to produce the mature protein; lane 2: βGPP+GiiscU+serine and cysteine protease inhibitors showing no inhibition; lane 3: βGPP+GiiscU+EDTA showing inhibition of cleavage; lane 4: Mutant βGPP in which E37 was mutated to glutamine+GiiscU, showing that the mutation of a key residue for βMPP activity also eliminates βGPP activity.

Mitosomal and some hydrogenosomal targeting presequences lack distal positively charged residues

To further investigate the structure-function relationships of the GPP, HPP and MPP, we screened in silico the G. intestinalis and T. vaginalis proteomes for putative mitosomal and hydrogenosomal N-terminal presequences ( Tables S1 and S2), which were then analyzed for structural elements known to mediate substrate-MPP interactions. In particular, we searched for the positively charged residues proximal to the cleavage site (P ₂ or P ₃), and those which are N-terminally distal from the processing site. The distance between the proximal and distal group was defined to be at least 3 amino acid residues [18],[19]. Giardia mitosomal presequences were predicted in three of nine putative mitosomal proteins ( Table S2). All of these presequences possess the proximal P ₂ arginine within a conserved cleavage motif [(ARV)R(F/L)(L/I)T], but the distal positively charged residues are absent ( Table S2). The lengths of the Giardia mitosomal presequences that have been experimentally verified are 10, 12 and 18 amino acid residues. The majority of the in silico predicted Trichomonas hydrogenosomal presequences (147) resemble the Giardia pattern; having a length of 4 to 21 amino acid residues, possessing a P ₂ arginine within a cleavage motif, and lacking the distal positively charged residues. However, we also detected 79 putative hydrogenosomal presequences, of 10 to 24 amino acids, that-like classic mitochondrial sequences-do contain distal arginines or lysines at position P ₆-P ₂₂.

Properties of MPP, GPP and HPP reflect the character of their respective substrates

To compare the specificities of the βGPP, α/βHPP and yeast α/βMPP in vitro, we tested their activity on a selection of mitosomal, hydrogenosomal and mitochondrial substrates ( Fig. 2). The βGPP cleaved only its own mitosomal substrates. By contrast, the α/βHPP cleaved the hydrogenosomal presequences, and the presequences of mitosomal ferredoxin and two mitochondrial substrates. The yeast α/βMPP processed all of the mitochondrial substrates and the two mitochondrial-like hydrogenosomal substrates that possess distal positively charged residues. We also tested whether we could make chimeric peptidases using a combination of hydrogenosomal and mitochondrial subunits. Interestingly, while the yeast αMPP did not interact with the Trichomonas βHPP, Trichomonas αHPP was able to form a heterodimer with yeast βMPP. However, this heterodimer did not cleave mitochondrial or hydrogenosomal substrates under our experimental conditions (data not shown).

To gain further insights into the structure-function basis of their different substrate spectra, we modelled each of the different proteins ( Fig. 4 and Fig. S4), using the yeast MPP structure as a guide [10]. For yeast MPP, the substrate is first recognized by the glycine-rich loop of αMPP [6],[7] and then moved to the active site of βMPP which interacts with the substrate cleavage motif including the proximal arginine. The distal positive residues of the presequence help to stabilize the substrate-MPP complex by binding to negatively charged residues within the large polar cavity formed by the α/βMPP subunits [10]. The part of the substrate-binding cavity formed by βMPP thus displays an evenly distributed negative charge to accommodate both proximal and distal positively charged residues of mitochondrial presequences. The αMPP interacts only with the distal positive residues of longer (>20 amino acid residues) mitochondrial presequences [9],[10].

(A) Predicted charge polarity distribution of the βHPP subunit and βGPP based on the known structure and charge distribution of the Saccharomyces cerevisiae βMPP subunit [10]. Red and blue colours denote negative and positive charge (±5 kT/e where kT is thermal energy and e is unit charge), respectively, whereas white denote relatively non-polar regions. The yellow asterisk marks the Zn-binding region in the active site of the enzyme (shown in b). The negative charges are distributed evenly in the cavity of βMPP while in the cavity of βGPP the negative charges are concentrated mainly around the active site. The MODELLER program [29] version 9.2 was used to build 3-D models of αHPP, βHPP and βGPP. The electrostatic properties of the model were evaluated using APBS version 0.5.1 [33]. (B) Alignment of key segments where negatively charged residues of βMPP are located and known to interact with the substrate. Numbered residues are those of yeast βMPP. E ₁₆₀ and D ₁₆₄ make a salt bridge with substrate residue R-2 (P ₂) and F ₇₇ interacts with P ₁′ which is also often a F residue. H ₇₀-X-X-E ₇₃-H ₇₄ is the conserved motif of the active site.

As the βGPP functions as a monomer we predict that its substrates, including the proximal arginine, interact directly with the negatively charged region of its catalytic site ( Fig. 4). The rest of the predicted βGPP cavity is, unlike βMPP, positively charged, although its predicted overall fold structure still resembles that of βMPP ( Fig. S4). The difference in βGPP charge distribution is compatible with the absence of distal positively charged residues in the mitosomal presequences, and, along with the absence of an αMPP-like subunit, may explain the inability of βGPP to process mitochondrial-type presequences. The simplicity of GPP is consistent with the highly reduced function of mitosomes and likely reflects (i) the paucity of proteins that are targeted to this organelle when compared with mitochondria and (ii) lack of N-terminal cleavable presequences in the majority of mitosomal proteins, including βGPP itself. As shown above ( Table S2), only nine mitosomal proteins have been identified so far and these are involved either in organelle biogenesis (Gipam18, GiHsp70, GiCpn60, GPP) or the formation of Fe-S clusters (GiiscS, GiiscU, GiiscA, Gigrx, Gifdx), which is currently the only known mitosomal function for G. intestinalis. Of these, seven are targeted to mitosomes in the absence of a detectable N-terminal targeting signal and thus function independently of GPP. Other than these, no other homologues of mitochondrial proteins have so far been identified in the genome of G. intestinalis [13].

The T. vaginalis HPP represents an intermediate stage between GPP and MPP in terms of charge distribution and enzymatic activity. Thus, it can process presequences with- or without distal positive residues, but can only cleave the shorter mitochondrial presequences ( Fig. 2). The presence of mitochondrial type presequences on hydrogenosomal proteins is consistent with the retention of the αHPP, which is likely involved in their recognition via its glycine-rich loop and/or their docking at the cleavage site.

Our phylogenetic and functional analyses show that the Giardia GPP is a striking example of reductive evolution from a heterodimeric to a monomeric enzyme, with properties resembling the putative ancestral α-proteobacterial enzyme, rather than the highly specialized MPP heterodimer found in well characterized mitochondria. While the principal selective pressure for the evolution of the processing peptidases is probably their ability to efficiently process substrates, the differences in the properties of the substrate presequences may also reflect the mode of their translocation across the organelle membranes [20]. In mitochondria from species across the phylogenetic tree [21], the positive residues of N-terminal presequences are recognized by the outer membrane TOM system and then the inner membrane translocase complex TIM23 [3]. Interestingly, no receptors (Tom20, Tom 22, Tom70) or components of the translocation channel of the TOM complex (Tom40, Tom5, Tom6, Tom7) have so far been identified for G. intestinalis [13] or T. vaginalis [22]. Putative core components of the TIM23 translocase (Tim23, Tim17) as well as Pam18 involved in protein transfer to the matrix have been found in T. vaginalis, but only Pam18 was found in G. intestinalis [14]. It thus appears that reductive evolution of the organelles has dramatically affected both the processing peptidases and the protein import pathway [21], with important implications for general models of mitochondrial biosynthesis, structure and function.

Materials and Methods

Phylogenetic analysis

Complete sequences of βGPP, βHPP, and βMPP were aligned with Muscle [23] to calculate sequence identity and similarity values. MPP, GPP and HPP sequences were aligned with Muscle [23] and analysed with Gblocks [24] to remove ambiguously aligned sites. Bayesian phylogenetic analyses were conducted using P ₄ ( http://www.nhm.ac.uk/research-curation/projects/P4/index.html). The optimal substitution model for Bayesian analyses was identified by ProtTest [25] (WAG+Gamma), a polytomy prior [26], and one or more base composition vectors, which were free to vary during the chain under the NDCH model [17]. MCMC chains were run for 1,000,000 generations, sampling trees and parameters every 200 generations. Model parameter proposal tuning values were determined using the P ₄ "autoTune" method. The burn-in was identified using the method of Beiko and co-workers [27]. The base composition component of the model was tested by simulation of the base composition χ2 statistic [17] at each sampling point, resulting in a posterior predictive distribution [28] against which the statistic of the original data could be tested using tail-area probability. Composition vectors were successively added until adequate fitting of the observed data to the model was identified (see Fig. S1).

Preparation of recombinant proteases and substrate proteins

The βGPP (NCBI accession: XP_001707100), αHPP (XP_001276882) and βHPP (XP_001316822) subunits and their substrates were expressed with hexahistidine tags in E. coli. An α/βHPP heterodimer was assembled from βHPP-His and non-tagged αHPP subunits by incubation of lysates of E. coli expressing the respective proteins for 30 min on ice in 20 mM Tris, 20 mM NaCl (pH 8.6), 1 mM MnCl ₂. All recombinant proteins were purified by nickel column chromatography (HiTrap Chelating) under native (βGPP-His, αHPP-His, βHPP-His, and α/βHPP-His) or denaturing (substrate proteins) conditions. An α/βMPP heterodimer was prepared as published [18].

In vitro protease activity assays

The GPP reactions were carried out in 20 mM Tris (pH 8.0), 100 mM NaCl, 1mM MnCl ₂, 30 min at 37°C, the HPP reactions in 20 mM Tris-HCl (pH 8.6), 20 mM NaCl, 2 mM MnCl ₂, 30 min at 37°C and activity of MPP was determined in 50 mM HEPES (pH 7.4), 20 mM NaCl, 1 mM MnCl ₂, 30 min at 30°C. To identify the cleavage sites, all substrates processed by the three proteases were subjected to N-terminal protein sequencing by Edman degradation. The kinetics of GPP was determined using the method published by Arretz and co-workers [8]. For determination of the activity of the HPP subunits, purified αHPP-His and βHPP-His were incubated on ice for 30 min either alone, or mixed together with 1 mM MnCl ₂. After addition of TviscU, the reaction was allowed to proceed at 37°C for 60 min. The specific activity of HPP with a fluorescent substrate based on the presequence of TvAK [Abz-MLST LAKRF AY(NO ₂)GKKDRM] (Bachem, Switzerland) was measured at 420 nm, with an excitation wavelength of 315 nm (Infinite M200, Tecan).

Size exclusion chromatography of purified GPP

A pre-calibrated Superdex 200 column was used to determine the molecular mass of E. coli produced GPP, under native conditions. Affinity purified βGPP-His in buffer of 50 mM CHES (pH 9.5), 150 mM NaCl was loaded on the column and washed (0.5 ml/min), collecting 1 ml fractions. Protein-containing fractions were assayed for βGPP activity.

Sucrose gradient centrifugation of a Giardia mitosome-enriched fraction

The molecular mass of GPP expressed in G. intestinalis with a hemagglutinin (HA) tag was estimated under native conditions by sucrose gradient centrifugation [15]. The mitosome-enriched fraction was isolated from a G. intestinalis homogenate using a published method [14]. The proteins in the mitosomal-enriched fraction were then separated on a calibrated sucrose gradient [15]. Fractions were analysed by immunoblot using anti-HA antibodies. Bands visualized by alkaline phosphatase were quantified by densitometry (GS-800 Calibrated Densitometer, BioRad).

Hydrogenosomal and mitosomal presequence identification

An application based on the NetBeans Platform ( http://platform.netbeans.org) was developed to search for proteins containing N-terminal hydrogenosomal and mitosomal presequences in the predicted T. vaginalis ( http://www.trichdb.org/trichdb/) and G. intestinalis ( http://www.giardiadb.org/giardiadb/) proteomes, respectively. Hydrogenosmal presequences were predicted based on two main parameters extracted from 21 known hydrogenosomal presequences: (i) the cleavage site motif, specified as RXF/(ILFSAGQ) or R(FNESG)/(ILFSAGQ) (the slash indicates the cleavage site and brackets mean one residue position), and the presequence start motif defined as ML(STACGR) or MTL or MSL. In addition, tryptophan was forbidden from the presequence, the maximum presequence length was optimized to 25 residues. Any presequences with overall negative charges were excluded (the approximate presequence charge at pH7 was counted according to the Henderson-Hasselbalch equation using the following pKa values: N-terminus 8.0, lysine 10.0, arginine 12.0, histidine 6.5, glutamic acid 4.4, aspartic acid 4.4, tyrosine 10.0, and cysteine 8.5). The G. intestinalis proteome was searched for N-terminal presequences based on three experimentally verified mitosomal presequences of known mitosomal proteins [13] ( Table S2). The parameters defined for the search were as follows: the cleavage site motif was defined as R(FS)/(IL)T, the presequence start motif as M(SLT), the maximum presequence length was set up to 20 residues, tryptophan was forbidden from the presequence. A search using parameters for prediction of hydrogenosomal presequences did not reveal additional mitosomal protein candidates.

Protein structure prediction

The MODELLER program [29] version 9.2 was used to build 3-D models of αHPP, βHPP and βGPP. Alignments of the βGPP and βHPP with the βMPP (pdbid 1HR6) [10] and of the αHPP with the αMPP (pdbid 1HR6) [10] were carried out using the PROBCONS web service [30] and manually edited. The quality of the final model was checked using the ProCheck [31] and WhatCheck [32] programs. The electrostatic properties of the model were evaluated using APBS version 0.5.1 [33].

Supporting Information

Bayesian model composition fit to the data assessed by posterior predictive simulation. Bars show the posterior distribution of χ2 for the homogeneous composition model (red) and the heterogeneous composition (NDCH) model with 10 composition vectors (green) in comparison to the statistic from the observed data. The simulated data for the NDCH model include the χ2 statistic from the observed data whereas the simulated data from the homogeneous model do not, the NDCH model thus provides a much better fit to the data. The original χ2 statistic for the data was 1292. In the simulations from the homogeneous analysis, this statistic ranged between 617 and 933 (mean = 763), while in the heterogeneous analysis (10 composition vectors) the statistic ranged between 877 and 1487 (mean = 1132 ).
https://doi.org/10.1371/journal.ppat.1000243.s001
(0.08 MB PDF)

Bayesian phylogenetic analysis of MPP-like protein sequences using the NDCH model [17] that allows for across-tree changes in protein amino acid composition. The tree is a majority rule consensus of 3,500 trees sampled from the posterior probability distribution of an MCMC with 10 across-tree composition vectors. Scale bar indicates estimated substitutions per site. Values on branches are posterior probabilities. Bacterial MPP homologues are shown in black, αMPP in red and βMPP in blue. Trichomonas α- and βHPPs and Giardia βGPP are highlighted in green.
https://doi.org/10.1371/journal.ppat.1000243.s002
(0.51 MB PDF)

The enzyme kinetics of the monomeric βGPP. The Lineweaver-Burk double reciprocal plot of reaction velocity, calculated as concentration of processed GiiscU in µM per minute versus concentration of GiiscU precursor. The least square fit line through the data intercepts x and y axes at −1/K m and 1/V max, respectively. The kinetic parameters calculated for βGPP were: V max = 1.7 µM/ min; K m = 8.4 µM; kcat = 17 min −1.
https://doi.org/10.1371/journal.ppat.1000243.s003
(0.10 MB PDF)

Tertiary structures of MPP, HPP and GPP. Homology models of βHPP and βGPP were built using the known structure of Saccharomyces cerevisiae βMPP, αHPP was modelled using S. cerevisiae αMPP. β-sheets are shown in yellow, α-helices in red, loops in grey. The glycine-rich loop of the α subunits and the zinc-biding motif of β subunits are highlighted in green. The MODELLER program [29] version 9.2 was used to build 3-D models of αHPP, βHPP and βGPP. The PROCHECK program version 3.5.4 was used to verify the validity of the model and gave a overall G-factor value of −0.12, which is well above −0.5; values below −0.5 indicates unusual structures [34]. Secondary structure prediction with PSIPRED [35] was also consistent with the modelled structure, recovering all five beta-sheets and the majority of alpha-helices (12 of 18).
https://doi.org/10.1371/journal.ppat.1000243.s004
(5.37 MB PDF)

Acknowledgments

We thank M. Marcincikova for technical support, Z. Voburka for protein N-terminal sequencing, J. Horvath for the development of the presequences identification software, and DeLano Scientific LLC for PyMol.

Author Contributions

Conceived and designed the experiments: TME JJ JT. Performed the experiments: OS AM TK IH EK. Analyzed the data: OS SRH MN LH RPH JT. Wrote the paper: OS TME JT.

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Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

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Author Summary In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called α or β, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis.

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Mitochondrial Type Iron-Sulfur Cluster Assembly in the Amitochondriate Eukaryotes Trichomonas vaginalis and Giardia intestinalis, as Indicated by the Phylogeny of IscS 

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Mitochondrial Type Iron-Sulfur Cluster Assembly in the Amitochondriate Eukaryotes Trichomonas vaginalis and Giardia intestinalis, as Indicated by the Phylogeny of IscS 

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https://academic.oup.com/mbe/article/18/10/1919/1060642?login=true

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Abstract

Pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.

Introduction

Amitochondriate eukaryotes can be divided into two metabolic types ( Martin and Müller 1998 ; Müller 1998 ). Type I organisms such as Giardia and Entamoeba lack organelles involved in core energy metabolism, while type II organisms (trichomonads, some ciliates, and chytrid fungi) harbor a double-membrane limited organelle, the hydrogenosome ( Müller 1993 ; Hackstein et al. 1999 ; Kulda 1999 ). The hydrogenosome is the site of the FeS protein-mediated metabolism of pyruvate and the formation of molecular hydrogen, which is accompanied by substrate-level phosphorylation ATP synthesis. In type I amitochondriates, the FeS protein-dependent pyruvate metabolism takes place in the cytosol ( Reeves 1984 ; Ellis et al. 1993 ). The evolutionary origin of amitochondriate eukaryotes is much debated. Although they were long regarded as ancestral premitochondrial lineages ( Cavalier-Smith 1987 ), recent evidence suggests that they had experienced the endosymbiotic event leading to the establishment of the mitochondrion ( Embley and Hirt 1998 ; Martin and Müller 1998 ; Roger 1999 ). In type II organisms, hydrogenosomes are regarded as descendants of a common endosymbiont that evolved to either mitochondria or hydrogenosomes ( Martin and Müller 1998 ; Müller 1997 ). A common origin of the two organelles is supported by a number of similarities in their structure, function, and biogenesis ( Johnson, Lahti, and Bradley 1993 ; Benchimol, Johnson, and De Souza 1996 ; Bui, Bradley, and Johnson 1996 ; Bradley et al. 1997 ; Dyall et al. 2000 ), as well as by phylogenetic analysis of several hydrogenosomal metabolic enzymes ( Länge, Rozario, and Müller 1994 ; Hrdý and Müller 1995a, 1995b ) and heat shock proteins ( Müller 1997 ; Embley and Hirt 1998 ). Although neither mitochondria nor hydrogenosomes have been found in type I organisms, genes of probable mitochondrial origin have been identified in Giardia ( Roger et al. 1998 ) and Entamoeba ( Clark and Roger 1995 ). Moreover, a putative mitochondrial "remnant," the mitosome ( Tovar, Fischer, and Clark 1999 ) or crypton ( Mai et al. 1999 ), has recently been detected in Entamoeba ( Müller 2000 ). These considerations led us to the hypothesis that FeS proteins operating in the energy metabolism of mitochondrial as well as secondarily amitochondrial organisms were present in the common ancestral organelle. If so, a common mechanism of FeS cluster assembly may operate in amitochondriate and in mitochondrial eukaryotes.

In spite of the importance of FeS proteins for all living cells, little is known of how and where FeS clusters are synthesized in vivo and which proteins are involved in their insertion into the apoproteins. The best-characterized enzyme participating in this process is a pyridoxal-5′-phosphate-dependent cysteine desulfurase which catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. Initially, the enzyme was described as NifS in Azotobacter vinelandii ( Zheng et al. 1993 ), in which it provides sulfur for FeS cluster formation in nitrogenase ( Zheng and Dean 1994 ). Later, a NifS homolog designated IscS (iron-sulfur cluster) was found in A. vinelandii, as well as in a number of non-nitrogen-fixing bacteria. It has been proposed that IscS plays a general role in the formation of FeS clusters or repair of FeS proteins with a housekeeping function ( Zheng et al. 1998 ). More recently, a major role of IscS in de novo FeS cluster synthesis has been demonstrated using an iscS deletion strain of Escherichia coli ( Schwartz et al. 2000 ). Importantly, IscS homologs have been identified in the genomes of diverse eukaryotes ( Arabidopsis, Caenorhabditis, Drosophila, Homo, Mus, Saccharomyces), suggesting a general role for IscS in FeS cluster formation. In mice ( Nakai et al. 1998 ) and yeast ( Strain et al. 1998 ), IscS is localized in mitochondria. In human cells, the IscS homologs are targeted either to mitochondria or to the cytosol and nucleus ( Land and Rouault 1998 ). Mutation in an iscS-like gene in yeast ( NFS1) caused reduction in the activities of the mitochondrial FeS proteins, aconitase and succinate dehydrogenase ( Strain et al. 1998 ). According to Kispal et al. (1999) and Lill and Kispal (2000) , mitochondria also play a crucial role in the FeS cluster formation of extramitochondrial FeS proteins. In addition, IscS homologs have been found to mediate several other functions that are independent of FeS cluster assembly but require IscS as a sulfur donor. Thus, IscS homologs are involved in biosynthesis of thiamin ( Lauhon and Kambampati 2000 ), NAD ( Sun and Setlow 1993 ), 4-thiouridine ( Kambampati and Lauhon 1999 ), and molybdopterin ( Amrani et al. 2000 ). Finally, IscS/NifS homologs mediate release of elemental selenium from L-selenocysteine ( Mihara et al. 2000 ), and they may participate in tRNA splicing ( Kolman and Söll 1993 ).

IscS has not been reported in amitochondriate eukaryotes, although FeS proteins are of particular importance for these organisms ( Müller 1998 ). Here we report the identification of genes encoding IscS in the type II organism Trichomonas vaginalis and the type I organism Giardia intestinalis. Phylogenetic analysis indicates the presence of a common mechanism for FeS cluster formation in mitochondria and hydrogenosomes, as well as in organisms that secondarily lost the mitochondrion/hydrogenosome-like organelles.

Materials and Methods

Organisms and Genomic DNA Preparation

Trichomonas vaginalis strain NIH-C1 (ATCC 30001) and G. intestinalis strain WB, clone 6 (ATCC 300957), were used. Genomic DNA was isolated from T. vaginalis using a guanidium thiocyanate procedure ( Wang and Wang 1985 ) and from G. intestinalis using a Blood & Culture DNA kit (Qiagen, Chatsworth, Calif.).

Probe Preparation, Cloning, and Screening of the Genomic Library

To obtain probes for screening a T. vaginalis genomic library, two pairs of degenerate primers, a GC-rich one and an AT-rich one, were designed based on the conserved regions of IscS/NifS sequences in GenBank (National Center for Biomedical Information): EIIFTSGATE (GC-rich sense: 5′-GARATYATYTTCTCVTCHGGHGCHACHGAR-3′; AT-rich sense: 5′-GAAATWATWTTYACWWSWGGWGCWACWGAA) and HKIH/YGPKGV/IG (GC-rich antisense: 5′-CCRAYDCCYTTTGGDCCRTRRATYTTRTG-3′; AT-rich antisense: 5′-CCWAYWCCYTT TGGWCCRTRWATYTTRTG-3′). Corresponding fragments were amplified by PCR, purified with a gel extraction kit (Qiagen) and cloned into pCR 2.1 vector (TA cloning kit, Invitrogen). The inserts were excised from the vector, gel-purified, and labeled by means of a Random Primers DNA Labeling System (GIBCO/BRL) with α-[ ³² P]dATP. These probes were used for screening a genomic DNA library in λ ZAP II vector (Stratagene). The sequences of positive clones were determined for both strands by primer walking.

Nucleotide sequences of Escherichia coli and Saccharomyces cerevisiae IscS/ NifS homolog genes were used to search the Giardia lamblia genome sequence database ( http://www.mbl.edu/baypaul/Giardia-HTML/index2.html; McArthur et al. 2000 ) with the BLAST program. Clones Ai0824 and Ki1686 contained sequences homologous to the N- and C-terminal ends of the bacterial and eukaryotic homologs. Based on the nucleotide sequence of these clones, we designed a pair of oligonuclueotide primers (sense: 5′-GATGACGAGCGTGCAAGGAAAGCTC-3′; antisense: 5′-GGTGACTACATGCGGATGCTCAGCC-3′) located in the 5′ and 3′ untranslated regions of the putative G. intestinalis nifS homolog gene, respectively. PCR reactions, utilizing these oligonucleotides and G. intestinalis genomic DNA as template, amplified a 2.4-kb fragment that was purified, cloned into the pCR2.1 vector (Invitrogen), and sequenced.

Sequence Alignment

Nucleotide and protein database searches were performed at the National Center for Biomedical Information using the BLAST program ( Altschul et al. 1997 ). Sequences were extracted from databases using the BlastAli program ( http://www.joern-lewin.de/). The IscS sequences of T. vaginalis and G. intestinalis were aligned to sequences from 64 taxa using ClustalX ( Thompson et al. 2000 ). The alignment was further edited visually with the use of the ED program of MUST ( Philippe 1993 ). The alignment of all 67 taxa resulted in 231 shared amino acid positions, while an alignment of 21 selected taxa consisted of 362 shared amino acid positions. The T. vaginalis TviscS-1, T. vaginalis TviscS-2, and G. intestinalis GiiscS sequences have been submitted to GenBank under accession numbers AF321005, AF321006, and AF311744, respectively.

Phylogenetic Analysis

Phylogenetic relationships were analyzed by means of the Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods using PHYLIP, version 3.6 ( Felsenstein 1989 ), and by the Maximum-Likelihood (ML) method using the PROTML program in MOLPHY, version 2.3 ( Adachi and Hasegawa 1996 ). The ML tree was constructed by local rearrangement of an NJ tree using the Jones-Taylor-Thornton model of amino acid substitutions with the F-option (JTT-F) to account for amino acid frequencies in the data set. User-defined trees were analyzed to compare alternative topologies ( Kishino and Hasegawa 1989 ). The local bootstrap proportion value was calculated for each internal branch of the ML tree using a local rearrangement option of the PROTML program. Bootstrap support for distance and parsimony analyses were based on 100 resampled data sets using SEQBOOT, PHYLIP, version 3.6.

Results and Discussion

Analysis of T. vaginalis and G. intestinalis iscS Genes and Putative Translation Products

Sequences for two complete iscS genes from T. vaginalis ( TviscS-1 and TviscS-2) and for one gene from G. intestinalis ( GiiscS) were obtained. The T. vaginalis genomic DNA library was screened with two probes derived from PCR products. The products were amplified using the A+T-rich or the G+C-rich degenerate primer pairs. Each pair amplified a distinct DNA fragment of about 4.4 kb. The two products displayed only 60% nucleotide sequence identity. Both fragments were identified as IscS/NifS homologs by a BLAST search. These products used as probes recognized separate sets of positive clones of the genomic library containing either TviscS-1 or TviscS-2 genes. No cross-reactivity between these clones was observed. The isolated clones contained complete putative open reading frames (ORFs) without intron-like sequences. The G+C content of TviscS-1 was rather low (39.7%), and that of TviscS-2 was higher (46.7%). The TviscS-1 and TviscS-2 coded for proteins 385 and 411 amino acids in length, respectively. Predicted molecular mass and an isoelectric point for the putative products for TviscS-1 were 41.9 and 8.2, while they were 44.8 and 8.2 for TviscS-2, respectively. The G. intestinalis gene isolated contained a 52% G+C-rich ORF coding for a polypeptide of 433 amino acids with a predicted molecular mass of 47.7 and an isoelectric point of 6.3.

Amino acid sequences deduced from the Trichomonas and Giardia genes were compared with IscS/NifS homologs from 64 species, including bacteria, fungi, plants, invertebrates, and vertebrates (alignment available on request from J.T.). An alignment of selected sequences including that of eubacterial IscS from A. vinelandii and mitochondrial sequence from S. cerevisiae is shown in figure 1 . Both Trichomonas and Giardia sequences contained all conserved regions proposed to mediate the cysteine desulfurase activity in IscS/NifS-like proteins: (1) His111 (numbered according to TviscS-1), which is involved in initial deprotonation of the substrate ( Kaiser et al. 2000 ); (2) the pyridoxal-5′-phosphate-binding site with the Schiff base forming Lys222 residue and Asp187 and Gln190, which bind the pyridine nitrogen and the phenolate oxygen of PLP, respectively, and residues involved in forming an additional six hydrogen bonds anchoring the phosphate group: Thr82, His221, Ser/Thr219, and Thr250 ( Zheng et al. 1993 ); and (3) the substrate-binding site including Cys371, which provides a reactive cysteinyl residue ( Zheng et al. 1994 ), as well as Arg362, Asn162, and Asn 41, which anchor the cysteine with a salt bridge and hydrogen bond ( Kaiser et al. 2000 ).

Importantly, the alignment revealed short N-terminal extensions of the trichomonad IscS sequences which were not present at the N-terminus of IscS in Giardia or in the eubacterial sequences. The PSORT II program ( http://psort.nibb.ac.jp/), designed for prediction of cleavage sites for mitochondrial presequences, recognized the SRS/YF motif with a characteristic arginine at position −2 relative to the cleavage site at the TviscS-2 N-terminus ( fig. 2 ). The TviscS-2 extension was typically serine-rich, consisting of eight amino acids, which started with leucine. It resembled N-terminal leader sequences found in proteins targeted to the Trichomonas hydrogenosome and can be assumed to have the same function ( fig. 2 ). Interestingly, TviscS-1 also started at the same position and contained an eight-amino-acid N-terminal extension; however, it did not contain the typical −2 arginine, and PSORT II did not recognize the consensus cleavage sequence. Nevertheless, a function of TviscS-1 presequence in organelle targeting cannot be ruled out, as a presequence without arginine has recently been reported for the hydrogenosomal membrane protein Hmp31 ( Dyall et al. 2000 ). No N-terminal extension of IscS was expected in Giardia, as this organism contains neither mitochondria nor hydrogenosomes. Lack of the N-terminal extension was previously reported in Giardia Hsp60, another mitochondrial-type protein recognized in this organism ( Roger 1998 ).

The presence of all key elements required for cysteine desulfurase activity in the deduced proteins suggests that IscS homologs are involved in Fe-S cluster assembly in T. vaginalis and G. intestinalis. The presence of the conserved N-terminal leader sequence in TviscS-2 suggests that in trichomonads the IscS-like proteins are targeted to hydrogenosomes, which are the likely sites of Fe-S cluster assembly. In Giardia, the FeS clusters are possibly assembled in cytosol.

A C-terminal sequence signature differentiates proteobacterial and eukaryotic IscS from homologs in all other organisms. TviscS-2 and GiiscS also contain this signature, which consists of 20 to 21 amino acids with consensus sequence SPL(W/Y)(E/D)(M/L)X(K/Q)XG(I/V)D(L/I)XX(I/V)XWXXX ( fig. 1 ). NifS genes of nitrogen-fixing bacteria also possess a similar C-terminal extension that starts with the Ser-Pro motif, but the subsequent sequence is not conserved. The only sequence from a eukaryote that lacks this signature is TviscS-1. The lack of this signature and the "atypical" N-terminal extension might indicate that Tvisc-1 is a pseudogene or that its product has a different function or localization. Eukaryotic IscS is distinguished from all other organisms, including proteobacteria, by the invariable Cys113 in the substrate deprotonation region. This residue is present in both trichomonad iscS products as well as in GiiscS. Prokaryotes possess Ala, Ser, or Gly at this position. Interestingly, Giardia IscS possessed two unique highly hydrophilic inserts, Thr137-Glu145 and Glu300-Ser321, which are not present in any of the 66 other species. It will be of interest to determine the function of such inserts, which might be associated with a specific localization of the gene product in Giardia.

Our sequencing data provide a solid basis for the prediction of the function of the products of iscS genes in amitochondriate eukaryotes. However, further studies are required to confirm their physiological function and cell localization.

Phylogenetic Analysis of IscS/NifS-like Sequences

In all global phylogenetic reconstructions, IscS/NifS-like homologs formed two distinct groups that were previously designated groups I and II ( Mihara et al. 1997 ). The IscS sequences of Trichomonas and Giardia and those of the mitochondrial homologs in other eukaryotes formed a single clade (group I) with a high bootstrap value (99%) using the local rearrangement option of the PROTML program ( fig. 3 ). Within this clade, Trichomonas and Giardia formed a subgroup together with Plasmodium falciparum and Arabidopsis thaliana. The second eukaryotic subgroup consisted of metazoan IscS, and the third group comprised homologs in fungal mitochondria. The α-proteobacterium Rickettsia prowazeki, often considered a close relative to the mitochondrial ancestor, clustered together with metazoan mitochondrial IscS ( Andersson and Kurland 1999 ).

The IscS of proteobacteria formed a sister group to eukaryotes. It is known that nitrogen-fixing bacteria possess two separate gene clusters that are involved in FeS cluster assembly: products of the nif gene cluster, including NifS, participate in nitrogenase-specific FeS cluster formation ( Zheng and Dean 1994 ), and isc gene products, including IscS, which have a more general function in the assembly of other Fe-S proteins ( Zheng et al. 1998 ). Our analysis suggests that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. The more specialized NifS sequences formed a distinct group with 99% local bootstrap support in the PROTML analysis which contained exclusively nitrogen-fixing proteobacteria.

Additional IscS/NifS-like homologs of eukaryotic organisms ( Mus musculus and A. thaliana) also appeared in another two distinct clades. The mouse homolog was located in a heterogeneous eubacterial group ( fig. 3 ). This gene codes for the cytosolic pyridoxal-5′-phosphate-dependent selenocysteine lyase, which resembles NifS in primary structure as well as in catalytic function ( Mihara et al. 2000 ). If the partial sequence of a human counterpart was also included in the analysis (data not shown), it was a sister group to the mouse sequence. The function of bacterial gene products of this clade has not been studied except in Synechocystis sp. (S74526 corresponds to sll0704 in CyanoBase http://www.kauza.or.jp/cyano/ investigated by Kato et al. [2000] ) and Bacillus subtilis (AAA21613 corresponds to the nifS-like gene according to Sun and Setlow [1993] ). The product of the Synechocystis gene showed selenocysteine lyase activity, although it also acted on L-cysteine sulfinic acid and other substrates ( Kato et al. 2000 ). The B. subtilis gene product has been suggested to participate in NAD biosynthesis ( Sun and Setlow 1993 ). Thus, it is likely that other members of this clade also have biochemical functions that are different from those of NifS and IscS proteins. The topology of A. thaliana genes was of particular interest. While one gene was related to the subtree of genes coding for mitochondrial IscS in protists, a second gene was placed in group II together with Synechocystis S76601. Since cyanobacteria share a common ancestor with plastids, we analyzed the second A. thaliana sequence for its possible subcellular localization with the PSORT program. The analysis gave the highest score for a chloroplast stroma localization (certainty = 0.501) of the gene product. This analysis suggests that the second IscS homolog of A. thaliana may operate in the chloroplast. However, it is difficult to predict its possible function. Group II consists of the most divergent NifS/IscS homologs. Function has been established only for products of two E. coli genes, which encode cysteine sulfinate desulfinase (F65063; Mihara et al. 1997 ), and for selenocysteine lyase (H64925; Fujii et al. 2000 ). Thus, the A. thaliana IscS homolog, as well as other members of this heterogenous bacterial group, might have functions different from FeS cluster formation, and their distance from genes of group I might reflect different evolutionary pressures. In any case, further biochemical studies on the functions of group II members are required.

The global gene tree showed that amitochondrial and mitochondrial IscSs share a common eubacterial ancestor, suggesting a common biosynthetic mechanism for FeS proteins. The tree also showed that both eukaryotes and bacteria possess several paralogous or orthologous IscS/NifS-like genes. The topology of these gene trees possibly reflects their specialized function or cell localization more than their large-scale phylogenetic relationships. Therefore, in a subsequent analysis we restricted the data set to eukaryotic and proteobacterial IscSs. The trees constructed by the ML, MP, and NJ methods confirmed the close relationship between amitochondrial and mitochondrial IscS with high bootstrap support ( fig. 4 ). The robustness of the relationship within the eukaryotic group was further assessed through the analysis of alternative tree topologies. We defined six branches on the tree: (1) amitochondriates and Plasmodium, (2) Arabidopsis, (3) Fungi, (4) Metazoa, (5) Rickettsia, and (6) Proteobacteria. Evaluation of the 105 alternative trees confirmed a common ancestry of genes from amitochondriate and mitochondriate organisms. However, the branching order within the eukaryotic clade was not resolved, as several alternative positions for eukaryotic subtrees with comparable significance were found ( table 1 ). We further suspected that the subtree of protists could be affected by long-branch attraction in spite of high bootstrap support. Thus, the positions of the Trichomonas and Giardia genes were tested using a data set from which we removed the most divergent sequences of A. thaliana and P. falciparum. The analysis clearly showed the instability of the protist group. Although NJ and MP reconstruction placed Trichomonas and Giardia as sister taxa with bootstrap support of 85% and 87%, respectively, ML constraint analysis gave comparable support to several alternative hypotheses. In the best tree topology and in four other tree topologies with Δln L <1 SE from the best tree, Trichomonas and Giardia were not placed as sister taxa ( table 1 ). Nevertheless, in all alternative trees, both amitochondriates were part of the eukaryotic clade.

Our results indicate that a common mechanism mediates FeS cluster assembly in mitochondriate and amitochondriate eukaryotes, even though different sets of FeS proteins function in these organisms ( Müller 1998 ). Both mitochondriate and amitochondriate eukaryotes require FeS proteins for single-electron transport processes associated with ATP production linked to the utilization of oxygen (mitochondriates) or not (type I and II amitochondriates). In mitochondria, FeS proteins are involved in the respiratory chain (subunits of complexes I, II, and III) and in the citric acid cycle (aconitase), while low-redox-potential FeS proteins such as pyruvate : ferredoxin oxidoreductase and ferredoxin are present in the hydrogenosomes of type I and in the cytosol of type II amitochondriates. Hydrogenosomes also contain hydrogenase. The presence of a common machinery responsible for FeS cluster formation and incorporation into "aerobic" and "ananerobic" types of apoproteins is supported by (1) the identification of the genes encoding IscS, a key member of machinery involved in FeS cluster assembly, in T. vaginalis and G. intestinalis, and (2) the placement of IscS of these amitochondrial organisms within the eukaryotic clade in phylogenetic analysis. In addition, a partial sequence of IscU, another protein involved in FeS cluster formation, has been identified in the database of the Giardia genome project ( http://www.mbl.edu/baypaul/Giardia-HTML/index2.html).

A common origin of FeS cluster formation in mitochondriate and amitochondriate eukaryotes could be explained by the recently proposed hydrogen hypothesis of eukaryotic origin ( Martin and Müller 1998 ). The hypothesis assumes that all eukaryotes, including contemporary amitochondrial organisms, once harbored the mitochondrion/hydrogenosome-like organelle derived from a proteobacterial endosymbiont. The ancestral endosymbiont is viewed as a facultatively anaerobic proteobacterium which possessed both anaerobic and aerobic metabolic machineries for electron transport-linked ATP production, including "aerobic" and "anaerobic" types of FeS proteins. A possible scenario is that the "anaerobic" set of FeS proteins was preserved in hydogenosomes, whereas the "aerobic" set was preserved in mitochondria. Both organelles inherited the common mechanism of the FeS cluster assembly. This scenario is supported by the close phylogenetic relationship between eukaryotic and proteobacterial IscS proteins. Our hypothesis is also congruent with a common origin of mitochondria and Trichomonas hydrogenosomes, as well as a secondary loss of the mitochondrion/hydrogenosome-like organelle in Giardia. We cannot, however, rule out alternative explanations for the origin of hydrogenosomes and biochemistry of amitochondrial eukaryotes, including the mechanism of FeS cluster assembly. Indeed, independent lateral gene transfers ( Doolittle 1998 ) or preservation of certain biochemical pathways from an anaerobic past of eukaryotic evolution might be involved. Nevertheless, comparative analysis of mechanisms responsible for the formation of FeS clusters, which are considered to be among the most ancient biologically active metal cofactors ( Cammack 1996 ), appears to be a promising tool for tracing eukaryotic history.

We thank Dr. Tetsuo Hashimoto for discussions and critical reading of the manuscript and Dr. Gang Wu for encouragement and help. We also thank Dr. Mitchell L. Sogin and Dr. Andrew G. McArthur for the use of the Giardia genome database. Oligonucleotide synthesis and DNA sequencing were performed at the Protein/DNA Technology Center at Rockefeller University. This work was supported by National Institutes of Health grant AI11942 to M.M. and by a grant from the Grant Agency of the Czech Republic to J.T. (204/00/1561). A major part of this work was performed during J.T.'s stay at the New York laboratory, supported by the NATO Science Fellowships Program.

References

# data[].attributes.attachment_link.title
Mitochondrial Type Iron-Sulfur Cluster Assembly in the Amitochondriate Eukaryotes Trichomonas vaginalis and Giardia intestinalis, as Indicated by the Phylogeny of IscS

# data[].attributes.attachment_link.description
Pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS).

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Mitocondria se escribe con M de misterio

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Mitocondria se escribe con M de misterio

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https://spark.siue.edu/bio_fac/126/
Romero Jr, A. (1976). Mitocondria se escribe con M de misterio.

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Title

Mitocondria se escribe con M de misterio.

Authors

Aldemaro Romero Jr., Southern Illinois University Edwardsville Follow

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Article

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1-1-1976

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Romero, A. 1976a. Mitocondria se escribe con M de misterio. Algo (289):23-25.

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Romero, Aldemaro Jr., "Mitocondria se escribe con M de misterio." (1976). Biological Sciences Faculty Research, Scholarship, and Creative Activity. 126.
https://spark.siue.edu/bio_fac/126

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Mitocondria se escribe con M de misterio.

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By Aldemaro Romero Jr., Published on 01/01/76

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Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle

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Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle

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https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-021-01129-7

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Research article

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BMC Biology 19, Article number: 193 (2021) Cite this article

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Abstract

Background

The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes.

Results

We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells.

Conclusion

Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

Introduction

Mitochondria are indispensable for producing most cellular ATP and providing other metabolites such as amino acids, lipids, and iron-containing prosthetic groups like heme and iron-sulfur (FeS) clusters [ 1]. Upon changing cellular conditions, their morphology undergoes constant re-arrangements known as mitochondrial dynamics, through which the cell controls metabolic capacity and the health of its key organelles [ 2]. Mitochondria either fuse to generate intricate networks throughout the cytoplasm or divide to form discrete organelles which more resemble their bacterial ancestors [ 3]. At the center of molecular machinery mediating the mitochondrial dynamics are dynamin-related proteins (DRPs) [ 4], but imperfect mitochondria are removed from the fusion-division cycle via specialized autophagy pathway [ 5]. Typical animal cells carry one to two thousand mitochondria which are stochastically segregated into two daughter cells during the cell division [ 6].

Studies on mitochondrial dynamics across different supergroups of eukaryotes showed that the extensive ongoing cycles of division and fusion are rather derived behavior of mitochondria not seen outside animal and fungal cellular models. This specifically concerns mitochondrial fusion which is either absent [ 7] or rely on unknown molecular machinery [ 8].

From the evolutionary perspective, it is likely that the early eukaryote contained a single mitochondrion that divided in synchrony with the cell as often observed for the bacterial endosymbionts of current eukaryotes [ 9]. Some eukaryotes have preserved this original blueprint of a single mitochondrion [ 10, 11, 12, 13] which divides in synchrony with the entire cell. However, only in case of kinetoplastids, the fundamentals of the synchrony were uncovered to some molecular detail [ 14, 15, 16, 17, 18]. Herein, so-called Tripartite Attachment Complex (TAC) physically connects basal body to the mitochondrial (mt) genome organized into a structure known as kinetoplast [ 17]. Specific TAC components were characterized across the complex from the basal body side (e.g., BBA4) [ 19], over the mitochondrial membranes (e.g., TAC40, TAC60) [ 20, 21] to the mitochondrial matrix (TAC102) [ 22], and their function is to coordinate the segregation of the replicated mt genomes/kinetoplast.

Giardia intestinalis is an anaerobic unicellular eukaryote (protist) from the Metamonada group which encompasses only organisms adapted to anoxic environments [ 23]. Accordingly, their mitochondria have been highly reduced to hydrogenosomes or mitosomes which are still cordoned by a double membrane but lost most of the other mitochondrial attributes such as the genome and respiratory chain [ 24]. Giardia mitosomes are the most reduced form of mitochondria as they are about 50-200 nm in size, do not produce ATP, and harbor only single metabolic pathway of FeS cluster formation [ 25]. Interphase mitosomes are very steady organelles, which do not fuse. Strikingly, their division seems to occur only during mitosis [ 26, 27, 28]. Of about the total 40 mitosomes per cell, several organelles can be, without exception, found between the two Giardia nuclei [ 25, 29, 30, 31]. These observations indicated specific attachment of the mitosomes to the nuclei or basal bodies of eight Giardia flagella. However, the molecular and structural basis for the recruitment of the organelles to this particular region of the cell and its actual function remained unknown.

Here, we tried to elucidate the nature of the association and its biological consequences towards the mitosis and cytokinesis of Giardia. Regarding the minute character of mitosomes, we used STED and FIB/SEM tomography to demonstrate that mitosomes are, via specialized microtubular fiber, bound to the axonemes of the oldest pair of Giardia flagella. We show that mitosomal inheritance is controlled by flagellar maturation and segregation. Comparisons with other Metamonada species suggest that the connecting fiber is a more common structure and perhaps one of the ancestral mechanisms to control mitochondrial segregation. Altogether, we demonstrate that the inheritance of the minimalist and genomeless mitochondrial organelles is under a careful control of the eukaryotic cell.

Results

Pre-existing central mitosomes segregate during prophase towards the poles of the mitotic spindle

All active cells of Giardia (trophozoites) contain two different populations of mitosomes, described as central and peripheral, which occur between two Giardia nuclei or are distributed all over the cytoplasm, respectively (Fig. 1A) [ 28, 29, 32]. While the latter predominantly associate with the tubules of the endoplasmic reticulum (ER) [ 28], the central mitosomes form a short array of several adjacent organelles between two Giardia nuclei which stay extremely steady until the initiation of mitosis [ 25, 31, 33]. Both the central and the peripheral mitosomes seem to be functionally identical as all mitosomal proteins detected by specific antibodies or by overexpression of the tagged proteins localize to both populations. All mitosomes, central and peripheral, divide during mitosis [ 28]. During early prophase, a rod-like array of mitosomes develops into a transient V-shape assembly which indicates a division or segregation of the organelles towards the opposite sides of the mitotic spindle [ 28]. To test if two sets of central mitosomes are formed during mitosis, we followed their behavior in live. To this aim, we took advantage of recently developed enzymatic Yellow Fluorescence-Activating and absorption-Shifting (Y-FAST) tag which outperforms GFP and related fluorescent proteins in anoxic environments [ 34]. The culture of Giardia cells expressing the fusion of mitosomal targeting presequence of GrpE with Y-FAST was enriched with the mitotic cells (see the " Methods" section). The cells were mounted to 0.5% agarose and observed in live in the presence of HMBR substrate. Upon focusing on the plane of the central mitosomes, we were able to detect rare events of the partitioning of the fluorescent signal corresponding to central mitosomes (Fig. 1B, Additional file 1: Movie S1). We estimated that the partitioning occurred during a 12-s window. As an alternate approach, we compared the size of the fluorescent signal of the central mitosomes in the fixed interphase cells and mitotic cells undergoing early prophase (Fig. 1C). The calculated values were 1.47 ± 0.25 μm and 0.90 ± 0.22 μm for the interphase and the mitotic cells (each n = 50), respectively. This supported the split of the fluorescent signal corresponding to the central mitosomes and further suggested that either the segregation of pre-existing mitosomes or the actual division of the organelles occurs during mitosis.

Partitioning of Giardia central mitosomes during mitosis. A The schematic drawing of Giardia trophozoite depicts central and peripheral mitosomes (red). The first are found between two nuclei, where also basal bodies and axonemes of Giardia flagella are localized. The latter are scattered across cytoplasm. There are four pairs of flagella called according to their position in the cell as anterolateral (light blue), posterolateral (dark green), ventral (light green), and caudal (dark blue). Additionally, the helical sheet of microtubules forms the adhesive disc of Giardia (gray). Immunofluorescence microscopy image of mitosomes labeled by anti- GL50803_9296 antibody (red), anti-β-tubulin. Merged image on the right includes nuclei stained with DAPI. The inlet is DIC image of the cell. B Giardia expressing the fusion of targeting presequence of mitosomal GrpE and Y-FAST were enriched for mitotic cells using albendazole treatment and incubated with 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) substrate. The reversible binding of Y-FAST to the fluorogenic substrate induces its green fluorescence (530-540 nm) upon excitation at 470-480 nm. The observed plane was focused on the central mitosomes. The series of images demonstrates fast partitioning of the fluorescence corresponding to central mitosomes. Red arrowheads highlight the position of central mitosomes. C The size of the fluorescent signal corresponding to the central mitosomes in the interphase and mitotic cells (the averaged values of 30 cells are shown). The scale bars 2 μm.

Mitosomes associate with the axonemes of Giardia flagella

The stable position of the central mitosomes between two Giardia nuclei together with the segregation of two mitosome populations during the onset of mitosis indicated some sort of physical association with the intracytoplasmic axonemes or basal bodies of flagella. Giardia basal bodies were suggested to act as microtubule organizing centers (MTOCs) of the mitotic spindle as they become a part of the spindle poles during the re-orientation of two nuclei [ 35, 36, 37]. To experimentally test the presence of the physical linkage between mitosomes and flagellar cytoskeleton, the cells were gently lysed by the passage through a hypodermic 33G needle (see the " Methods" section). The lysed cells were fixed and mitosomes and microtubules labeled by specific antibodies (Fig. 2A). Indeed, the lysed cells showed an intact association of the central mitosomes with the axonemes, even when the rest of the cell cytoplasm was removed during the sample preparation. In contrast, there were no peripheral mitosomes observed. Furthermore, the samples were prepared for 2D stimulated emission depletion (STED) microscopy (Fig. 2B) which revealed that in the partially disturbed bundle of the axonemes, mitosomes can be found associated only with some of the axonemes. Alternate 3D-DyMIN STED approach [ 38] was used to image the region on more intact samples from the transverse plane (Fig. 2C, D). Here, the central mitosomes were found surrounded by axonemes, right at the center of their bundle (Fig. 2E, Additional file 2: Movie S2 and Additional file 3: Movie S3). These results suggested that an unknown connector between the axonemes and the central mitosomes is responsible for the organelle segregation during cell division.

Central mitosomes associate with the flagellar axonemes. A Giardia cells were gently lysed by the passage through 33G needle, and the ruptured cells were analyzed by immunofluorescence microscopy. The immunolabeling of β-tubulin (green) and mitosomal marker GL50803_9296 (red) revealed that the intact mastigonts bear attached central mitosomes, while the cytoplasmic membrane and rest of cytoplasm was removed during lysis, scale bar 2 μm. Upper and bottom panels represent two examples of the same situation. B 2D STED superresolution microscopy of the mastigonts with associated mitosomes (primary antibodies as in A), scale bar 0.2 μm (0.1 μm in the insets), 30 nm resolution in XY axes. Upper and bottom panels represent two examples of the same situation. C The central mitosomes imaged in the frontal plane, scale bar 2 μm, and in D the transverse plane by 3D STED superresolution microscopy (primary antibodies as in A). Four optical sections are shown, scale bar 0.4 μm; the resolution was 100 nm in Z-axis and 120 nm in XY plane. E The reconstruction of 3D STED by Imaris illustrates the position of mitosomes (red) among eight axonemes (green), sections shown in D are indicated. C, A, P - caudal, anterolateral and posterolateral flagella, respectively.

Additional file 3: Movie S3. Movie of Imaris reconstruction of central mitosomes built on 3D STED.

Imaging of the mitosomes using FIB/SEM tomography

While the confocal and superresolution microscopy provided experimental support for the presence of a molecular bridge or a cytoskeletal connector between the central mitosomes and the axonemes, the nature and the architecture of the connection remained entirely unknown. Hence, the high-resolution imaging of the entire Giardia cells by electron microscopy seemed essential to obtain such structural details. Specifically, focused ion beam scanning electron microscopy (FIB/SEM) was employed as it enables to reconstruct spatial arrangement of cell structures upon rendering the information of sequential data sets. To this aim, Giardia cells were flat embedded (Additional file 4: Fig. S1) [ 39] and, using the correlative approach, the interphase and mitotic cells were selected for FIB/SEM analysis (Additional file 4: Fig. S1, see the " Methods" section). The cells were processed by customized rOTO protocol and analyzed by fine milling (voxel size 3 × 3 × 8 nm) [ 39, 40] (Fig. 3A, B, Additional file 5: Fig. S2). The 3D reconstruction of the mitosomes throughout the entire cell volume showed that in addition to the axonemes, approximately one half of the mitosomes (central and peripheral) is in connection to other cellular structures such as the ER, the adhesive disc, and lysosome-like compartments known as peripheral vacuoles [ 41, 42, 43] (Fig. 3C, D). Regularly, there was only one contact with other compartments detected per mitosome. Finally, clusters of several (peripheral) mitosomes could be found in the cytosol (Fig. 3C). In general, the association of mitosomes with cellular compartments was comparable between interphase and mitotic cells (Fig. 3D). The inspection of the mitosomal interior revealed further compartmentalization/segmentation (Fig. 4A, C), suggesting that the two mitosomal membranes do not align entirely and the inner membrane establishes tiny mitosomal subcompartments. The previous EM images of the mitosomes revealed non-spherical prolonged shape of the organelles [ 25, 32]. In addition, we noticed that mitosomes share one flattened side, i.e., contain flat regions of double membrane (Fig. 3A-C, Fig. 4 C). This shape was common to mitosomes from all parts of the cell with different orientation to the cell axis concluding that the flattening is an intrinsic property of the organelles not caused by sample preparation or neighboring cell structures.

FIB/SEM imaging of mitosomes reveals contacts with other cellular compartments. A The SEM imaging was optimized to enable visualization of the mitosomes. The examples of central and peripheral organelles are shown. They all share asymmetric, semi-oval shape with one flattened side of the organelle. B The volume rendering (volren) of several SEM layers increases the resolution of the imaging of the cell interior. N-nucleus, white arrowhead-mitosome, red star-axoneme, white star-basal body. C The mitosomes were found to associate with several cellular compartments, i.e., the axonemes (AXO), endoplasmic reticulum (ER), adhesive disc (disc), other mitosomes (mito-mito), and the peripheral vacuoles (PV). The red arrowheads point to visible connection. D The distribution of the contacts of mitosomes with other cellular compartments in the interphase and the mitotic Giardia ( n = 48 and n = 106 organelles, respectively)

The mitosomes are segmented and share the asymmetric shape with one flattened side. A Mitosomes show internal compartmentalization. The examples of different size and architecture of the organelles are shown. B Statistics of the mitosomal dimensions show conserved organelle shape ( n = 30 and n = 70 organelles in the interphase and mitotic cells, respectively). C Almost all mitosomes have flattened shape and segmented interior ( n = 48 and n = 106 organelles in the interphase and mitotic cells, respectively). D The estimated mitosomal volume ( n = 17 organelles)

In order to describe this shape quantitatively, the longest dimension (length) and the dimension perpendicular to it (width) were measured and plotted (Fig. 4B). The both dimensions averaging around 150 nm and 75 nm, respectively, were found to be very conserved across mitosomes, hence suggesting that the mitosomal size and shape are defined by the intrinsic protein and lipid composition. In addition, vast majority of the organelles contained membrane indentations forming possible additional subcompartments (Fig. 4C, E). Finally, we used 3D reconstructions of 17 mitosomes to estimate that the average mitosomal volume is around 0.0011μm ³ (Fig. 4D). In the context of the estimated volume of the entire Giardia trophozoite to be 440 μm ³, all (forty) mitosomes represent only a fraction of 0.00025% of the whole cell volume.

Central mitosomes are connected to only the eldest (caudal) pair of flagella via a microtubular connector

The examination of the region of interest between two Giardia nuclei by FIB/SEM tomography was facilitated by the characteristic pattern of axonemes of the eight Giardia flagella (Fig. 5A). There are four distinct pairs of flagella named according to positions at which they emerge from the cell as ventral, posterolateral, anterolateral and caudal (Fig. 1A). Importantly, during each cellular division, the flagella undergo a flagellar maturation cycle in which two caudal flagella represent the eldest pair of the flagellar cycle [ 36].

FIB/SEM analysis reveals connecting microtubular fiber between central mitosomes and the axonemes of caudal flagella. A The frontal plane image of the central mitosomes of interphase Giardia between the longitudinally sectioned axonemes as reconstructed from the transversal FIB/SEM images, AXO-axoneme, red arrowhead-microtubular fiber, N-nuclei. B The illustrative transversal section shows two fibers (red arrowheads) connecting central mitosomes and axonemes of caudal flagella. M-mitosomes. C The reconstruction of the FIB/SEM analysis depicting the overall architecture of the microtubular fiber (yellow) between central mitosomes (red) and axonemes of caudal flagella (dark blue). Axonemes of anterolateral flagella are shown in light blue, N-nuclei. D The dimensions of the connecting fiber ( n = 15 and n = 3 for the length and of the connector and the binding region, respectively). E Transmission electron micrographs show the detailed structure of the connecting microtubular fibers, which each consists of 3 to 4 microtubules. M-mitosomes

In the FIB/SEM micrographs of the region of tightly packed axonemes between Giardia nuclei, a population of several central mitosomes could be seen, showing a typical array of the organelles as observed by fluorescence microscopy (Fig. 5A). The number of central mitosomes was slightly variable with the average number to be eight in the interphase cells ( n = 5). Detailed analysis of the individual micrographs revealed a direct linear connection between each of the central mitosomes and always one of the two neighboring axonemes (Fig. 5A, B). Interestingly, only two of eight axonemes were involved in mitosome binding and the overall reconstruction revealed that these correspond to the pair of caudal flagella (Fig. 5C). However, it could not be resolved, if a specific microtubule doublet is involved in the binding. The length of the connector (distance between the mitosome and the axoneme) was estimated to be around 90 nm (Fig. 5D), while the actual mitosome-binding region of the connector in the anteroposterior axis was approx. 1.1 μm (Fig. 5D. The connector itself accompanied the whole length of the intracytoplasmatic portion of caudal flagella (Fig. 5 C).

To get higher resolution of the connector, Giardia cells were examined by transmission electron microscopy (TEM). While TEM was not optimal for mitosome investigation, it nicely exposed the structural features of the connector as a sheet of microtubules-a microtubular fiber (Fig. 5E). Hence, we could conclude that the central mitosomes are specifically linked via microtubular fiber to the axonemes of the caudal flagella. Further microscopic examination revealed that the mitosomal connector is integrated into the upper part of the so-called funis system of Giardia, which represents the cytoplasmic microtubular skeleton alongside the two caudal flagella (Additional file 6: Fig. S3) [ 44, 45].

Coordination of mitosomal inheritance and the flagellar cycle

The data presented above demonstrated that mitosomes segregate during mitosis to the opposite ends of the mitotic spindle probably via the link to caudal flagella. This has raised a fundamental question on the coordination of mitochondrial inheritance and the flagellar cycle, during which a new caudal flagellum is established by the transformation of one (mitosome-free) anterolateral flagellum. Of particular importance was (i) at what stage the connecting microtubular fiber develops on the axoneme of new caudal flagellum, (ii) when the central mitosomes are acquired, and (iii) whether the new central mitosomes are derived from the pre-existing organelles linked to the old caudal flagellum.

To get appropriate answers, different stages of mitotic Giardia were examined by FIB/SEM tomography. The flagellar re-arrangement starts at prophase when basal bodies/axonemes of caudal flagella deflect to the left and right side to set up the future sites of daughter cells morphogenesis. This is accompanied by a profound reorientation of anterolateral flagella axonemes (Fig. 6), which migrate to the opposite direction and each join the other/one caudal flagellum at the opposite side of the cell [ 36]. At that time the central mitosomes split into two populations, each alongside one caudal flagellum (Fig. 6). At the re-oriented anterolateral flagellum, a new tiny connecting fiber (150-300 nm in length) is being formed, however, at this stage, without attached mitosomes (Fig. 6).

The orchestrated mitosomal segregation and flagellar maturation during mitosis. 3D reconstructions of mitosomal and flagellar dynamics during mitosis shows (i) separation of the caudal flagella with the connected mitosomes during prophase. Note also the formation of the new connecting microtubular fiber on both anterolateral flagella. (ii) During meta/anaphase the mitosomes become associated with the new fiber on the former anterolateral flagella. (iii) During telophase, two new pairs of caudal flagella (the coloring according to the original interphase cell) are formed with more mitosomes (green) connected to the new caudal flagellum. AL1, AL2-anterolateral flagella; C1, C2-caudal flagella

During metaphase and anaphase, the fibers at caudal flagella acquire a new extended vaulted microtubular part with the distal curled end (Fig. 7). The accompanying mitosomes, however, remain associated at the end of the linear part of the fiber as seen in interphase and prophase. The number of mitosomes attached to caudal flagellum varies at this stage from 1 up to 6 with typically 4 organelles ( n = 6). The re-oriented anterolateral flagellum which joined the caudal flagellum transforms into a new caudal flagellum. The transformation is characterized by an apparent growth of the accompanying microtubular fiber, to which mitosomes are recruited (Fig. 6). At this stage, the shape of the fiber fully resembles the one of the old caudal flagellum.

The development of connecting microtubular fiber during mitosis. During the mitotic stages, the microtubular fiber extends and points towards the newly built axoneme. New extended vaulted microtubular part grows towards the newly assembled flagellar axonemes of daughter ventral and posterolateral flagella (light gray axoneme) and the fiber curls around a single microtubule (red)

During telophase, both old and new caudal (i.e., transformed anterolateral) axonemes have already reached their positions at the anterior of the future daughter cell. The elongation of the microtubular fiber reaches up to 3 μm in length. Interestingly, the vaulted extension of the fiber is oriented towards the newly assembled flagellar axonemes of daughter ventral and posterolateral flagella and may provide morphogenetic signals for oriented intracytoplasmic flagellar growth (Fig. 7). Strikingly, at this stage, the mitosomal populations at the old and new caudal flagella are of similar size (4-6 mitosomes), strongly indicating that the new caudal flagellum might recruit its mitosomes from the surrounding peripheral organelles (Additional file 7: Fig. S4). Finally, the closer pairing of the axonemes of two caudal flagella guides the two mitosome populations together (Fig. 6, Additional file 8: Movie S4). The two microtubular fibers accompanying caudal flagella become each a ventral and a dorsal part, respectively of the daughter cell funis (Additional file 5: Fig. S2).

Additional file 8: Movie S4. Animation of 3D reconstruction. Caudal flagella in dark blue, anterolateral flagella in light blue. The old and new connecting fibrils shown in yellow and white, respectively. Central mitosomes shown in red.

Segregation of mitosomes occurs also when mitosis is aborted

The discovery of the tight association of Giardia mitosomes with the flagella and the orchestration of mitosome segregation during mitosis prompted us to investigate the impact of mitosis on mitosome dynamics. Giardia cells were incubated with albendazole which is a potent inhibitor of newly polymerized microtubules. The affected Giardia cells retain functional microtubular structures but are unable to assemble new microtubules such as those composing the mitotic spindle [ 46]. Cells visually undergoing cytokinesis were analyzed by fluorescence microscopy and compared to untreated control (Fig. 8A, B). Immunolabeling of mitosomes and microtubules along with DAPI staining showed several distinguishable phenotypes (Fig. 8) [ 46], which were classified according to the number of nuclei (N) and the number of the flagellar apparatuses with the associated structures (M). While the untreated population contained almost exclusively interphase stage trophozoites with two nuclei and one flagellar apparatus (2N1M) and a small number (less than 1%) of mitotic cells (4N2M) (Fig. 8A, C), four different phenotypes could be distinguished in the albendazole treated cell culture (Fig. 8B). The most cells had two (undivided) nuclei and two separated flagellar apparatuses (2N2M), indicating that they did not complete cytokinesis (Fig. 8B, C). Three other phenotypes included cells with 0N1M and 2N1M arrangements and the least abundant 1N1M arrangement (Fig. 8B, C). There was also a small population of unaffected interphase cells with two nuclei and one flagellar apparatus (2N1M). In all cases, regardless of the number of cell nuclei, each flagellar apparatus co-localized with the fluorescent signal corresponding to the central mitosomes (Fig. 8 A, B). The inhibition of mitosis hence did not impact the segregation of the central mitosomes, which remained associated with the caudal flagella by the existing microtubules. This result showed that it is exclusively the flagella and not the nuclei that have control over the segregation of central mitosomes to the daughter cells.

Segregation of mitosomes occurs also when mitosis is impaired. The inhibition of the microtubule assembly results into the formation of aberrant cells with impaired nuclear segregation. A Vast majority of cells in untreated cell population are in the interphase stage containing two nuclei (N) and one flagellar apparatus (M) (2N1M). Less than 1% of cells undergo mitosis that ends with a cell carrying 4 nuclei and 2 flagellar apparatuses (4N2M). B Four cell types can be detected in the cell population classified according to the number of the nuclei and the flagellar apparatuses. The cell type 2N2M is pre-cytokinesis; the other cell types represent cells which underwent cytokinesis. Examples of the aberrant morphologies are shown. Immunolabeling by anti-β-tubulin antibody (green), mitosomes by anti-GL50803_9296 antibody (red). Nuclei are stained with DAPI (blue). The central mitosomes highlighted by white arrowheads. C The quantification of morphologies in albendazole treated ( n = 334 cells) and untreated ( n = 350 cells) populations

Discussion

The eukaryotic cell requires its mitochondria to be faithfully inherited by the daughter cells during the cell division. Being semiautonomous organelles with their genome and translation machinery, mitochondria propagate only from the pre-existing organelles [ 2, 47, 48]. Our current understanding of the history of the eukaryotic cell infers that its formation coincided with the origin of its mitochondrion [ 24], and hence, the mitochondrial inheritance has been an integral part of eukaryotic cellular functions from its early days.

Mitochondria of Opisthokonta (animals and fungi) have served as the principal models for studying mitochondrial dynamics and inheritance, e.g., [ 49, 50, 51]. Moreover, multiple health and developmental problems have been related to the erroneous dynamics of human mitochondria [ 51, 52, 53]. Herein, hundreds of mitochondria constantly fuse and divide to control the quality of the organelle and its genome with the assistance of the ER [ 54] and actin cytoskeleton [ 55]. The inheritance of the dynamic animal mitochondrial network thus occurs rather stochastically, even though there seems to be also cell cycle-dependent aspects of mitochondrial dynamics [ 6, 56, 57].

In contrast, there are large groups of eukaryotes like Kinetoplastida or Apicomplexa, which entirely harnessed the mitochondrial inheritance with their cell cycle [ 14, 15, 16, 17, 58, 59, 60]. These unicellular eukaryotes carry only a single mitochondrion, which thus must be carefully propagated to the daughter cells. Kinetoplastida have evolved a structure called tripartite attachment complex (TAC), which physically connects the mitochondrial genome with the basal body of its flagellum [ 17]. The segregation of the basal body is thus necessary for the partitioning of the duplicated mitochondrial genome (kinetoplast), which is quickly followed by nuclear division and cytokinesis [ 14]. In addition, a prominent microtubule quartet extending along the length of the cell plays a key role in the coordinated biogenesis of different organelles during the cell cycle [ 61].

Apicomplexa evolved schizogony, a synchronized cell division resembling intracellular budding, during which mitochondrial filaments enter into the emerging daughter cells [ 60, 62].

Eukaryotes from the supergroup of Metamonada (Fig. 9) [ 63] are all intriguingly adapted to inhabit anoxic or low oxygen environments. This has dramatically impacted the structure and function of their mitochondria. They all are genomeless and lack many mitochondrial functions including the respiratory chain and the coupled ATP synthesis. Due to their variability, these organelles are often referred to as mitochondria-related organelles (MROs) [ 71, 72, 73, 74]. Interestingly, MROs are always present in large numbers (in tens to hundreds), and concerning their dynamics, they are very stable during the cell cycle [ 7]. Yet, virtually nothing is known about how the organisms control their inheritance. Moreover, the genomeless organelles could in principle assemble de novo like the Golgi complex or the peroxisomes, e.g., [ 75].

Connecting the mitochondrial organelles to the flagellum may be an ancestral strategy of controlling the mitochondrial inheritance in Metamonada. The schematic tree of eukaryotic supergroups drawn according to [ 63] shows the position of Metamonada and within recently questioned Excavata supergroup. Bellow, a subtree of selected Metamonada species according to [ 64, 65]-the parasitic lineages are shown in red. Highlighted is the presence of the microtubular fiber or analogous structures (*) T. foetus [ 66], H. teleskopos [ 67], A. paluster [ 68], O. intestinalis [ 69], and S. muris [ 70]. Note the secondary absence of mitochondrial organelle in Monocercomonoides sp. (#)

We have thus studied the inheritance of mitosomes of Giardia, which are one of the simplest and smallest MROs known to date [ 25]. Mitosomes carry out only a single metabolic role in the formation of iron-sulfur clusters via the mitochondrial ISC pathway [ 25, 76]. Other metabolic hallmarks of mitochondria have been lost from mitosomes as expendable. However, despite their minimalism, mitosomes are still bounded by two membranes, which carry TOM and TIM complexes to import proteins from the cytosol [ 30, 31, 77]. Our data show that despite fulfilling the only fraction of the original mitochondrial functions, Giardia applies control over their inheritance. It does so by connecting a small subset of mitosomes to flagellar axonemes and specifically to those of the caudal flagella [ 36, 78]. From the perspective of the flagellar maturation cycle of Giardia, the caudal flagella represent the privileged flagellar pair. They are the eldest of four flagellar pairs, in which the left caudal flagellum (in dorsal view) being at the definitive position is considered as truly mature [ 36]. It serves as the organizing center for the assembly of the adhesive disc [ 79], which is resorbed during mitosis and re-built in daughter cells upon cytokinesis [ 80, 81] and for the initiation of the cytokinesis [ 82].

The examination of Giardia mitotic stages revealed that flagellar maturation involves the formation of mitosome-connecting microtubular fiber on the intracytoplasmic axoneme of the newly establishing (formerly anterolateral) caudal flagellum. During mitosis, the fiber rapidly elongates and accompanies the neighboring newly formed axoneme of ventral and posterolateral flagella. Hence, it is possible that the fiber also coordinates the architecture of daughter mastigonts before cytokinesis. The integration of mitosomal and flagellar inheritance was further supported by the experiment on albendazole-treated cells which were not able to undergo karyokinesis but proceeded into divided mastigonts and the central mitosomes.

At what stage do the central mitosomes associate with the newly formed fiber? Our observations strongly indicated that the central mitosomes become "central" only via the association with the connecting fiber as they were never found at this region of the cell just alone. It is noteworthy that also no mitosomal protein has been found exclusively in the central mitosomes [ 83]. Moreover, according to the most parsimonious scenario, new central mitosomes should be derived from the pre-existing central organelles, when the old caudal axoneme meets the new one. However, surprisingly, we observed mitosomes associated with the yet forming lamella on newly establishing caudal flagellum, before the full establishment of the caudal pair. This indicates that peripheral mitosomes might be able to "hop on" the microtubular fiber at the new caudal flagellum. This view is supported by observations of metaphase/anaphase cells with the already formed fiber at anterolateral flagella, however yet without mitosomes.

The inspection of the fiber beyond the region of the central mitosomes showed that it is likely a part of both the old and of the newly forming microtubular system of Giardia and other diplomonads known as funis, possibly R2 root in nomenclature of eukaryotic cytoskeleton [ 68], which runs in Giardia to the posterior end of the cell perhaps to anchor and relax the movement of the eight flagella [ 45, 69, 84].

To understand whether the microtubular fiber connecting MROs and the axonemes is a more universal cellular feature, we searched the available data on other Metamonada species outside the Giardia genus (Fig. 9). Strikingly, highly similar microtubular structure connecting MROs with the axoneme could be observed in the electron micrographs of Aduncisulcus palustris [ 68] and Hicanonectes teleskopos [ 67] which are free-living protists belonging to Carpediemonas-like organisms (CLOs) [ 85]. Together with Diplomonada where Giardia belongs, they constitute a larger Fornicata group within Metamonada [ 23]. Analogously, more distant eukaryotes from Parabasalia group like Tritrichomonas foetus are known to associate some of their MROs (hydrogenosomes) with the sheet of microtubules known as axostyle [ 66]. Hence, it is tempting to speculate that Metamonada at least partially control the inheritance of their genomeless MROs via the association with the stable microtubular cytoskeleton.

Unexpectedly, Giardia mitosomes were found to accommodate conserved asymmetric shape. The original discovery of Giardia mitosomes already showed the elongated central mitosomes [ 25]. While this could be explained as a shape imposed on the mitosomes by the neighboring axonemes, our FIB/SEM analysis has revealed that such morphology is also common to the peripheral organelles across the cytoplasm. The vast majority of mitosomes exhibit "bun" or a "bean" shape with opposite oval and flat sides (Fig. 4E). What may be behind such shape? It is well known that phospholipid bilayers spontaneously form spherical vesicles like many endomembrane vesicles or even some double membrane bounded MROs such as mitosomes of Entamoeba histolytica [ 86] or hydrogenosomes of Trichomonas vaginalis [ 87]. Other than the spherical shape of the membrane-bounded compartments thus reflects the presence of membrane proteins which affect the membrane flexibility [ 88]. In case of mitochondria, these can either be proteins that are specifically dedicated to membrane bending and folding such as OPA1/Mgm1 [ 89], proteins of MICOS components [ 90] or membrane proteins, whose primary role is unrelated to the compartment morphology, but their sole presence affects the membrane properties such as ATP synthase [ 91]. So far, no homologs of the actual mitochondrial morphology effectors have been identified in the Giardia genome, which could specifically explain the prolonged shape with flat double membrane arrangement. Thus, we can hypothesize that it is perhaps the different content of membrane proteins such as TOM and TIM complexes or so far unknown transport machinery, which stiffen the mitosomal membranes at certain regions. This internal organization occurs in one of the smallest mitochondrial compartments. Our measurements estimated that an average mitosome occupies 0.001 μm ³ and the whole population of the mitosomes takes up only 0.01% of the cell volume. This is in striking contrast to "classical" aerobic mitochondria of yeast or mammalian tissues which occupy 7% or even 35% of the cell volume, respectively [ 92, 93, 94], and nicely illustrates the overall reduction of the mitosomes and their relative contribution to cellular metabolism. However, despite being such minute compartments, they are carefully watched over by Giardia.

Conclusions

Our work demonstrates a unique type of mitochondrial inheritance which evolved in the anaerobic eukaryotes carrying highly reduced genomeless mitochondrial organelles. While we were able to describe the mechanism underlying the partitioning of the central mitosomes of Giardia, this work has raised other exciting questions on the relationship between the central and peripheral mitosomes and, more generally, on the evolutionary importance of the mitochondrion-flagellum connection. Does it represent the ancestral arrangement of the eukaryotic cell or rather a derived trait of the Metamonada group of protists? These questions will hopefully be answered in future studies.

Methods

Giardia intestinalis cultivation

G. intestinalis cells (strains WB, ATCC 30957 and HP-1- Prague line of Portland-1 isolate ATCC 3088) were cultured in TYI-S-33 medium supplemented with 10% heat-inactivated adult bovine serum (Sigma-Aldrich), 0.1% bovine bile, and appropriate antibiotics at 37 °C [ 95]. The enrichment of mitotic cells was done according to [ 36]. Trophozoites from late log phase were incubated in growth medium supplemented with 100 ng/ml of albendazole (Sigma-Aldrich) for 6 h at 37 °C [ 36]. After the incubation, the albendazole-affected detached cells were discarded, and the unaffected adherent pre-mitotic cells were washed twice with pre-warmed fresh drug-free medium and then detached from the tube by cooling on ice for 10 min. The cells were then allowed to proliferate on slides in the drug-free conditions for 9-20 min and fixed by ice-cold methanol (see below) for fluorescence microscopy or by 2.5% glutaraldehyde (see below) for electron microscopy. For the analysis of cells with blocked mitosis, the albendazole-affected (detached) cells were fixed by 1% formaldehyde (see below).

Plasmid construction and cloning

The first 300 base pairs of the gene GL50803_1376 were amplified from Giardia genomic DNA using specific primers: forward 5′-CATGCATATGGCGCTTTCTGCACTT-3′ and reverse 5′-CATGACGCGTGATCTCGTGGAGATGTTT-3′ containing NdeI and MluI restriction sites. After cleavage by NdeI and MluI restriction enzymes, the fragment was cloned into NdeI/MluI-linearized pTG plasmid [ 96]. The gene encoding Y-FAST protein was amplified from pAG87 plasmid (a kind gift from prof. Gautier, Sorbonne University, France) by forward 5′-CATG ACGCGTATGGAACATGTTGCC-3′ and reverse 5′-CATG GGGCCC TTATACCCTTTTGACAAACAC-3′ primers containing MluI and ApaI restriction sites, respectively. After cleavage with MluI and ApaI restriction enzymes, the fragment was cloned into MluI/ApaI-linearized pTG plasmid containing GL50803_1376 leader sequence.

Preparation of cells for fluorescence microscopy

For the immunofluorescence microscopy of the adherent cells, wild-type trophozoites were incubated on slides in TYI-S-33 medium for 15 min at 37 °C, fixed in ice-cold methanol for 5 min, and permeabilized in ice-cold acetone for 5 min. The blocking and the immunolabeling steps were all performed in a humid chamber using a solution of 0.25% BSA, 0.25% fish gelatine, and 0.05% Tween 20 in PBS for 1 h each. The cells were stained by an anti-GL50803_9296 antibody produced in rabbit (1:2000 dilution) [ 97]. The secondary antibody used was Alexa Fluor 488-conjugated anti-rabbit antibody (Life Technologies, 1:1000 dilution) or Alexa Fluor 594-conjugated anti-rabbit antibody (Life Technologies, 1:1000 dilution. After each immunolabeling step, the slides were washed three times for 5 min by PBS supplemented with 0.1% Tween20 (Sigma-Aldrich). Slides were mounted in Vectashield containing DAPI (Vector Laboratories).

In the case of detached cells that were affected by albendazole treatment, the cells were fixed by 1% formaldehyde for 30 min at 37 °C. The cells were then centrifuged at 900× g for 5 min at RT and washed in PEM buffer (200 mM PIPES, 2 mM EGTA, 0.2 mM MgSO ₄, pH 6.9). The cells were then resuspended in PEM buffer and transferred to poly-L-lysine-coated coverslips and let to attach for 15 min. The cells were then permeabilized by 0.2% Triton X-100 in PEM buffer for 20 min and washed three times with PEM buffer. Then, the cells were incubated in blocking solution PEMBALG (PEM supplemented with 1% BSA, 0.1% NaN ₃, 100 mM lysine and 0.5% gelatin) for 30 min. All blocking and immunolabeling steps were performed in humid chamber at room temperature. After blocking, the cells were incubated in PEMBALG containing anti-acetylated tubulin antibody produced in mouse (1:2000 dilution, Sigma-Aldrich) and anti-GL50803_9296 antibody produced in rabbit [ 97] (1:2000 dilution) for 1 h. The coverslips were then washed three times 15 min in 0.1% Tween-20 in PBS and incubated in PEMBALG containing anti-mouse antibody conjugated with Alexa488 (1:1000 dilution, Life Technologies) and anti-rabbit antibody conjugated with Alexa594 (1:000 dilution, Life Technologies) for 1 h. The coverslips were washed three times 15 min in 0.1% Tween-20 in PBS and mounted in Vectashield containing DAPI (Vector Laboratories).

For STED microscopy of gently lysed Giardia, the cells were resuspended in SM buffer supplemented with protease inhibitors (Complete, EDTA free, Roche) and passed 10 times through 33 × ½" needle (Cadence Inc.). The cell lysate was then put on Piranha-treated high-performance coverslip (Zeiss). After 15 min, the sample was fixed using 2% formaldehyde for 15 min. The cells were then permeabilized, blocked, and immunolabeled as described above. Secondary antibodies used in this experiment were as follows: anti-rabbit Abberior STAR 635p antibody (1:100 dilution, Abberior Instruments GmbH) and anti-mouse Abberior STAR 580 antibody (1:100 dilution, Abberior Instruments GmbH). Anti-fade liquid Abberior Mount (Abberior Instruments GmbH) was used for mounting the slides according to the manufacturer's protocol.

For live-cell imaging experiments, Giardia trophozoites expressing GiDHFR fused with Y-FAST tag [ 34] were enriched for mitotic cells as described above. Albendazole-unaffected cells were collected and placed to the 35 mm glass bottom microscopy dish (In Vitro Scientific). Cells were kept at 37 °C for 10 min to attach. After that, warm, fresh media supplemented with 1 μM HMBR (a kind gift from prof. Gautier, Sorbonne University, France) was added to the cells, and they were observed using Leica SP8 confocal microscope in 37 °C-preheated humid chamber (Okolab) under anaerobic conditions.

Fluorescence microscopy and imaging

Static images were acquired on Leica SP8 FLIM inverted confocal microscope equipped with 405 nm and white light (470-670 nm) lasers and FOV SP8 scanner using HC PL APO CS2 63x/1.4 NA oil-immersion objective. Laser wavelengths and intensities were controlled by a combination of AOTF and AOBS separately for each channel. Emitting fluorescence was captured by internal spectrally tunable HyD detectors. Imaging was controlled by the Leica LAS-X software. Images were deconvolved using SVI Huygens Professional software (Scientific Volume Imaging) with the CMLE algorithm. Maximum intensity projections and brightness/contrast corrections were performed in FIJI ImageJ software.

Live imaging experiments were performed on the same microscope as described above. Y-FAST fluorescence was excited at approximately 480 nm, and emitted light was approximately 540 nm as described in [ 34]. Fluorescence images were taken every 500 ms.

STED and 3D-DyMIN STED [ 38] microscopy were performed on a commercial Abberior STED 775 QUAD Scanning microscope (Abberior Instruments GmbH) equipped with Ti-E Nikon body, QUAD beam scanner, Easy3D STED Optics Module, and Nikon CFI Plan Apo 60x oil-immersion objective (NA 1.40). Samples were illuminated by pulsed 561 nm and 640 nm lasers and depleted by a pulsed 775 nm STED laser of 2D donut shape (all lasers: 40 MHz repetition rate). The fluorescence signal was detected with single-photon counting modules (Excelitas Technologies). Line-interleaved acquisition enabled separated detection of individual channels in the spectral range from 605 nm to 625 nm and from 650 nm to 720 nm. The confocal pinhole was set to 1 AU.

LM and CLEM of Giardia

For FIB/SEM experiments, laser marked slides were used (LASERMarking, Munich, Germany) and the cells in a proliferative phase were allowed to attach to them in a silicon anaerobic chamber at 37 °C. Slides were rinsed with PBS, pH 7.2, and immediately fixed with 2.5% (v/v) glutaraldehyde (Science Services GmbH, München) in 75 mM cacodylate (Sigma-Aldrich), 75 mM NaCl, 2 mM MgCl ₂ for 30 min, followed by 3 washing steps in cacodylate buffer. Cells were stained with DAPI, sealed with a coverslip and Fixogum (Marabu GmbH & Co. KG, Tamm, Germany) to prevent drying during LM investigation on Zeiss Axiophot fluorescence light microscope. The positions of cells in the desired stage of mitosis (ROIs) were marked on a template, with the same coordinates. For documentation, epifluorescence, phase contrast, and DIC images were taken in different magnifications (objective × 5, × 10, × 40), and these were sufficient to followingly retrieve the ROIs in SEM.

EM preparation

After removal of Fixogum and coverslip, cells were post-fixed (customized rOTO-impregnation protocol [ 39] with 1% (v/v) OsO ₄ and 1% (w/v) K ₄Fe(CN) ₆ in cacodylate buffer for 30 min, washed 3 times in ddH ₂O, incubated with 1% (w/v) thiocarbohydrazide in ddH ₂O for 30 min, washed = with ddH ₂O 3 times, followed by post-fixation with 1% OsO ₄ in ddH ₂O for 30 min. The samples were rinsed 3 times with ddH ₂ O and dehydrated in a graded series of acetone (20%, 40%, 60%, 80%, 100%, 100%, 100%), containing a 1% uranyl acetate step in 20% acetone for 30 min, infiltrated and embedded on the glass slide.

The ultrathin embedding was done according to [ 40] as follows. Cells were infiltrated with 1:1 Hard-Plus Resin-812 in acetone, 2:1 (resin/acetone) and 3:1 (resin/acetone) always for 1 h. Finally, the cells were placed in two changes of 100% resin, for 1 h and 2 h. A filter paper, completely soaked with acetone, was placed at the bottom of a Falcon® tube to provide an acetone saturated atmosphere. A polypropylene cap was placed on top of the filter paper to avoid direct contact with the slide. The slide was placed upright into the Falcon® tube, allowing the excessive resin to drain into filter paper at the bottom of the Falcon tube for 2 min. Then, the slides were centrifuged for 2 min, 268 g, Hettich, swing-out rotor). The samples were polymerized for 72 h at 60 °C. The size of the glass slides was reduced to appropriate size by fracturing with aid of a diamond pen. The specimens were mounted on an aluminum stubs with colloidal silver. A carbon coating by evaporation of approximately 15 nm thickness was done on Balzers BAE 080 T, Liechtenstein).

High-resolution FIB/SEM

Giardia interphase and mitotic trophozoites cells were imaged in a Zeiss Auriga 40 FIB/SEM workstation operating under SmartSEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). FIB/SEM milling was started right in front of the anterior part the cell. Ion beam currents of 50 pA were used. High-resolution images were obtained with the EsB detector at 1.5 kV at a grid voltage of − 500 V. Dependent on the desired resolution, image pixel sizes between 2 and 10 nm in x/y were chosen. The milling rate was set to 2 nm, which allows the adjustment of the z resolution in 2 nm steps at any time during the FIB/SEM run. The average voxel size achieved was 3 × 3 × 8 nm.

3D-reconstruction and visualization

The datasets were aligned using Amira (Thermo Fisher Scientific, USA) with the module align slices. The image stacks from FIB/SEM were segmented and reconstructed in Thermo Scientific Amira software or processed with a direct volume rendering algorithm (volren) for immediate visualization.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. The raw microscopy datasets are available from the corresponding authors on request.

References

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Acknowledgements

We especially thank prof. Jaroslav Kulda for his lifelong mentoring on the biology of Giardia intestinalis.

Funding

The project was supported by the Ministry of Education, Youth and Sports of CR (MSMT-10251/2019, project ID: LTAB19004 to P.T., by Bayerisch-Tschechische Hochschulagentur BTHA-JC2019-7 to A.K., by Czech Science Foundation Grant 20-25417S and the PRIMUS grant PRIMUS/SCV34 from Charles University to P.D., by PRIMUS/20/MED/008 from Charles University to P.T., by the National Sustainability Program II (Project BIOCEV-FAR, LQ1604) from the Ministry of Education, Youth and Sports of CR (MEYS), and by the project "Centre for research of pathogenicity and virulence of parasites" (No. CZ.02.1.01/0.0/0.0/16_019/0000759) funded by European Regional Development Fund. We acknowledge Imaging Methods Core Facility at BIOCEV, institution supported by the MEYS CR (Large RI Project LM2018129 Czech-BioImaging), and ERDF (project No. CZ.02.1.01/0.0/0.0/18_046/0016045) for their support with obtaining imaging data presented in this paper. We also acknowledge CMS-Biocev ("Biophysical techniques") of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127).

Author information

Authors and Affiliations

1. Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University, Prague, Czech Republic

Pavla Tůmová & Eva Nohýnková

2. Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic

Luboš Voleman & Pavel Doležal

3. Plant Development and Electron Microscopy, Department of Biology I, Biocenter of Ludwig-Maximilians University, Munich, Germany

Andreas Klingl

4. Department of Biology I, Biocenter of Ludwig-Maximilians University, Munich, Germany

Gerhard Wanner

Contributions

Conceived and designed the experiments: PT, LV, AK, EN, GW, PD. Performed the experiments: PT, LV, EN, GW. Analyzed the data: PT, LV, EN, AK, GW, PD. Contributed reagents/materials/analysis tools: PT, LV, EN, AK, GW, PD. Wrote the paper: PT, PD. All authors have read and approved the manuscript.

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Correspondence to Pavla Tůmová or Pavel Doležal.

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Tůmová, P., Voleman, L., Klingl, A. et al. Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle. BMC Biol 19, 193 (2021). https://doi.org/10.1186/s12915-021-01129-7
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DOI: https://doi.org/10.1186/s12915-021-01129-7

Keywords

Mitochondrial inheritance

Mitosomes

mitochondrial evolution

Flagellum

Cytoskeleton

Cell cycle

Mitochondrial division

Protist

Giardia

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Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle - BMC Biology

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Background The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study.

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

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

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https://www.nature.com/articles/nature01945

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Abstract

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.

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References

1. Hashimoto, T., Sánchez, L. B., Shirakura, T., Müller, M. & Hasegawa, M. Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proc. Natl Acad. Sci. USA 95, 6860-6865 (1998)

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3. Arisue, N., Sánchez, L. B., Weiss, L. M., Müller, M. & Hashimoto, T. Mitochondrial-type hsp70 genes of the amitochondriate protists Giardia intestinalis, Entamoeba histolytica and two microsporidians. Parasitol. Int. 51, 9-16 (2002)

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6. Riordan, C. E., Ault, J. G., Langreth, S. G. & Keithly, J. S. Cryptosporidium parvum Cpn60 targets a relict organelle. Curr. Genet. advance online publication, 20 August 2003 (doi:10.1007/s00294-003-0432-1)

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Acknowledgements

We thank G. Clark, J. Bowyer and S. Cutting for critically reading the manuscript. A recombinant plasmid containing the T. vaginalis ferredoxin gene was provided by J. P. Germanas and K. Krause. The use of partial genome sequence information from the Giardia Genome Project Database is acknowledged. The technical assistance of J. James and N. Sommerville is also acknowledged. M.H. is a sabbatical visitor supported by CINVESTAV, México. Research at the Rockefeller University (gene cloning, antibody generation) was supported by a NIH grant to M.M. Research at Charles University ( in vitro assembly of Fe-S clusters) was supported by a grant from FIRCA to J.Tachezy. J.M.L. (electron microscopy) was supported by a Research Leave Fellowship from the Wellcome Trust and by Tenovus Scotland. Research at Royal Holloway (bioinformatics, cell fractionation, fluorescence confocal microscopy, manuscript writing, project coordination) was supported by a Wellcome Trust grant to J.Tovar.

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Author notes

Lidya B Sánchez

Present address: Public Health Research Institute, Newark, New Jersey, 07103, USA

Authors and Affiliations

1. School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK

Jorge Tovar, Gloria León-Avila, Mark van der Giezen & Manuel Hernández

2. The Rockefeller University, 1230 York Avenue, New York, New York, 10021, USA

Lidya B Sánchez & Miklós Müller

3. Department of Parasitology, Faculty of Science, Charles University, Prague, 12844 2, Czech Republic

Robert Sutak & Jan Tachezy

4. School of Life Sciences, WTB/MSI complex, University of Dundee, Dundee, DD1 5EH, UK

John M. Lucocq

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Correspondence to Jorge Tovar.

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Tovar, J., León-Avila, G., Sánchez, L. et al. Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 426, 172-176 (2003). https://doi.org/10.1038/nature01945
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Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation - Nature

# data[].attributes.attachment_link.description
Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year1. 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 phylogenies1,2.