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See detailSubunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Colina-Tenorio, Lilia; Miranda-Astudillo, Hector; Cano-Estrada, Araceli et al

in Biochimica et biophysica acta (2016)

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes. [less ▲]

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See detailSubunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

in Biochimica et Biophysica Acta-Bioenergetics (2015), 1857(4), 359-369

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1 kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes. [less ▲]

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See detailEstudio de las interacciones de la subunidad atípica ASA1 en la ATP sintasa mitocondrial del alga Polytomella sp
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

Poster (2015, November)

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See detailDissecting the peripheral arm of the mitocondrial ATP synthase of chlorophycean algae.
Gonzalez-Halphen, Diego; Vazquez-Acevedo, Miriam; Miranda Astudillo, Héctor Vicente ULiege et al

Poster (2014, November 06)

Mitochondrial F 1 Fo-ATP synthase (complex V) makes ATP using the electrochemical proton gradient generated by the respiratory chain. It is an oligomeric complex embedded in the inner mitochondrial ... [more ▼]

Mitochondrial F 1 Fo-ATP synthase (complex V) makes ATP using the electrochemical proton gradient generated by the respiratory chain. It is an oligomeric complex embedded in the inner mitochondrial membrane that works like a rotary motor. Chlamydomonas reinhardtii and Polytomella sp., two members of the chlorophycean lineage of unicellular green algae, have a highly-stable dimeric mitochondrial F 1 Fo-ATP synthase, with an estimated molecular mass of 1600 kDa. The chlorophycean enzyme contains the eight conserved polypeptides present in the vast majority of eukaryotes that represent the main components of the proton-driven rotary motor and the catalytic sector of the enzyme: subunits alpha, beta, gamma, delta, epsilon, a (ATP6), c (ATP9), and OSCP. Nevertheless, and in sharp contrast with other mitochondrial F 1 Fo-ATP synthases like the one from beef heart, the algal enzyme seems to lack several classic components: the subunits of the peripheral stalk b, d, f, A6L, and F6, the subunits responsible for dimer formation e and g, and the regulatory polypeptide IF 1 . Instead, the algal enzyme contains nine subunits with molecular masses ranging from 8 to 60 kDa named ASA1 to ASA9 (for ATP Synthase Associated proteins). These polypeptides have no clear orthologs in the databases and seem to be unique to chlorophycean algae. The nine ASA subunits build up a highly-robust peripheral stalk with a unique architecture, as observed on single-particle electron microscopy (EM) images. Our group has found of interest to gain further insights on the close-neighbor relationships between the ASA subunits and their interactions with some of the classical subunits. We have therefore explored with some detail the topological disposition of the components of the algal mitochondrial ATP synthase using different experimental approaches: detection of subunit-subunit interactions based on cross-linking experiments, the yeast two hybrid system or reconstitution with recombinant subunits; generation of sub-complexes after partial dissociation of the dimeric ATP synthase; inference of subunit stoichiometry based on labelling of cysteine residues and modelling of the overall structural features of the complex from small-angle X ray scattering data and EM image reconstruction. Based on the results obtained from these diverse experimental strategies, we suggest a refined model for the topological disposition of the 17 polypeptides that constitute the algal mitochondrial ATP synthase. [less ▲]

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See detailStudy of the interactions of atypical subunit ASA1 in the ATP synthase of Polytomella sp.
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

Poster (2014, November 03)

The mitochondrial ATP synthase of chlorophycean algae has a structure different from that of other organisms. All the subunits that typically make up the the peripheral arm and those that are involved in ... [more ▼]

The mitochondrial ATP synthase of chlorophycean algae has a structure different from that of other organisms. All the subunits that typically make up the the peripheral arm and those that are involved in the dimerization of the enzyme are missing. In compensation, it has acquired nine subunits of unknown evolutionary origin that have been named ASA1 to ASA9. These ASA subunits are only present in chlorophycean algae and are not found in others closely related algal lineages, such as ulvophycean, prasynophycean and trebuxophycean green algae. Heat dissociation experiments, cross linking studies and electronic microscopy studies have allowed the proposal of a structural model of the ATP synthase of chlorophycean algae in which ASA subunits make up the peripheral arm and participate in the dimerization of the enzyme; however, the localization of ASA1 subunit remains unclear. The objective of this work is to clone and purify the ASA1 subunit to perform interaction studies in order to know which are its neighboring subunits and to propose its topological disposition in the peripheral arm of the ATP synthase of Polytomella sp. The experimental strategy is based on the cloning of the corresponding gene, the overexpression of the protein in Escherichia coli and the purification of the recombinant protein in order to perform interaction assays. In this work, we report an ASA1-OSCP interaction, which could link a classical protein of the enzyme, such as OSCP, with an atypical subunit unique to the chlorophycean algal lineage. [less ▲]

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See detailInteractions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Miranda-Astudillo, Hector; Cano-Estrada, Araceli; Vazquez-Acevedo, Miriam et al

in Biochimica et Biophysica Acta-Bioenergetics (2014), 1837

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See detailInteraciones de las subunidades Asa2, Asa4 y Asa7 en el brazo periférico de la ATP sintasa mitocondrial del alga incolora Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Conference (2013, October 23)

La F1Fo-ATP sintasa mitocondrial de las algas clorofíceas es un complejo parcialmente embebido en la membrana interna mitocondrial que se purifica como un dímero estable de 1600 kDa. Se encuentra formado ... [more ▼]

La F1Fo-ATP sintasa mitocondrial de las algas clorofíceas es un complejo parcialmente embebido en la membrana interna mitocondrial que se purifica como un dímero estable de 1600 kDa. Se encuentra formado por 17 polipéptidos, nueve de los cuales (subunidades Asa1 – Asa9) no se encuentran presentes en las ATP sintasas mitocondriales clásicas y parecen ser exclusivos de este grupo de algas. En particular, las subunidades Asa2, Asa4 y Asa7 parecen formar parte de una sección del brazo periférico de la enzima. En el presente trabajo se sobreexpresaron y purificaron las subunidades Asa2, Asa4, Asa7 y los correspondientes fragmentos amino terminal y carboxilo terminal de las subunidades Asa4 y Asa7, con el fin de realizar estudios de interacción in vitro, empleando técnicas inmunoquímicas, electroforesis azul nativa y cromatografía de afinidad. Se determinó que las subunidades Asa4 y Asa7 interaccionan fuertemente principalmente a través de sus regiones carboxilo terminal. Además, la subunidad Asa2 interacciona con Asa4 y Asa7 así como con la subunidad α en el sector F1. Las 3 subunidades Asa forman un subcomplejo Asa2/Asa4/Asa7 con una estequiometría 1:1:1. La subunidad Asa7 y el extremo carboxilo terminal de Asa4 parecen ser necesarios para la interacción con Asa2. Basados en los resultados se generaron modelos estructurales in silico de las tres subunidades. Se propone un modelo de la vecindad topológica de las tres subunidades (subcomplejo Asa2/Asa4/Asa7) así como de su posición en el brazo periférico de la ATP sintasa del alga. [less ▲]

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See detailEstudio de las interacciones in vitro de las subunidades que componen el brazo periférico de la ATP sintasa mitocondrial de Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Colina-Tenorio, Lilia et al

Poster (2012, November)

La ATP sintasa mitocondrial de las algas clorofíceas no contiene las subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas no contiene las subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima, tratamiento con agentes entrecruzadores y estudios de microscopía electrónica llevaron a la propuesta de un modelo estructural de esta ATP sintasa, en el cual las subunidades ASA1 a 9 forman la estructura del estator periférico y participan en la dimerización de la enzima (Cano-Estrada et al., 2010). El objetivo de este trabajo es conocer cómo interactúan las subunidades ASA2, ASA4 y ASA7 del brazo periférico de la ATP sintasa mitocondrial de Polytomella sp. La estrategia experimental está basada en la clonación de los genes completos así como de fragmentos de éstos, la sobre-expresión heteróloga en bacteria y su purificación. Con las proteínas purificadas se realizaron ensayos de interacción que comprenden inmunorréplicas tipo far western, geles azules nativos y copurificacion de subcomplejos en columnas de afinidad. Los resultados obtenidos indican una interacción entre las subunidades ASA4 y ASA7 mediada por los extremos carboxilo de ambas proteínas, así como una asociación de las subunidades ASA2 – ASA4 y ASA2 – ASA7. [less ▲]

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See detailAislamiento de algunas subunidades que integran el brazo periférico de la ATP sintasa mitocondrial de Polytomella sp. y estudio de sus interacciones.
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Conference (2011, November 14)

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima y tratamiento con agentes entrecruzadores llevaron a la propuesta de un modelo estructural de esta ATP sintasa, donde se propone que las subunidades ASA1 a 9 participan en la estructura del estator periférico y en la formación de un dímero estable. En el presente trabajo se desea abordar un estudio más detallado de las subunidades ASA, para conocer acerca de las interacciones que establecen entre ellas y como contribuyen a la formación del brazo periférico de la enzima. La estrategia experimental parte de la clonación de los genes de las subunidades en vectores de expresión en bacteria, la sobre-expresión de las subunidades recombinantes, su purificación y ensayos de interacción mediante geles azules nativos e inmunoréplicas tipo Far Western. Los resultados indican una fuerte interacción entre las subunidades ASA4 y ASA7 que involucra el extremo carboxilo terminal de la subunidad ASA4. Los estudios de inmunoréplica indican interacciones entre las subunidades ASA4-ASA7 y ASA4-ASA2. [less ▲]

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See detailInteracción de las subunidades ASA2, ASA4 y ASA7 de la ATP sintasa mitocondrial del alga incolora Polytomella sp
Cano-Estrada, Araceli; Miranda Astudillo, Héctor Vicente ULiege; Vázquez-Acevedo, Miriam et al

Poster (2011, November)

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See detailEstudio de las interacciones entre las subunidades ASA del brazo periférico de la ATP sintasa mitocondrial de Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Poster (2010, November)

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima, tratamiento con agentes entrecruzadores y estudios estructurales llevaron a la propuesta de un modelo estructural de esta ATP sintasa, donde se propone que las subunidades ASA1 a 9 participan en la estructura del estator periférico y en la dimerización de la misma. En el presente trabajo se desea abordar un estudio más detallado de las subunidades ASA, para conocer acerca de las interacciones que establecen entre ellas y como contribuyen a la formación del brazo periférico de la enzima. La estrategia experimental parte de la clonación de los genes de las subunidades en vectores de expresión en bacteria, la expresión de las subunidades recombinantes y su purificación. Los estudios de inmunoréplica indican las interacciones entre las subunidades ASA4-ASA7 y ASA4-ASA2. [less ▲]

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See detail3D-reconstruction and overall topology of the dimeric mitochondrial ATP synthase of the colorless alga Polytomella sp
González-Halphen, Diego; Vázquez-Acevedo, Myriam; Cano-Estrada, Araceli et al

in Biochimica et Biophysica Acta (BBA) - Bioenergetics (2010, July), 1797(Supplement 1), 32

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See detailSubunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp
Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam; Villavicencio-Queijeiro, Alexa et al

in Biochimica et Biophysica Acta-Bioenergetics (2010), 1797(8), 1439-1448

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that ... [more ▼]

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp. [less ▲]

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See detailThe fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp is dimeric
Villavicencio-Queijeiro, Alexa; Vazquez-Acevedo, Miriam; Cano-Estrada, Araceli et al

in Journal of Bioenergetics & Biomembranes (2009), 41(1), 1-13

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F-1 sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0-9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. [less ▲]

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See detailThe mitochondrial ATP synthase of chlorophycean algae contains eight subunits of unknown origin involved in the formation of an atypical stator-stalk and in the dimerization of the complex
Vazquez-Acevedo, Miriam; Cardol, Pierre ULiege; Cano-Estrada, Araceli et al

in Journal of Bioenergetics & Biomembranes (2006), 38(5-6), 271-282

Mitochondrial F1FO-ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator ... [more ▼]

Mitochondrial F1FO-ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator-stalk as well as those involved in the dimerization of the fungal and mammal complex. Instead, it contains eight novel polypeptides named ASA1 to 8. We show that homologs of these subunits are also present in the chlorophycean algae Polytomella sp. and Volvox carterii. Blue Native Gel Electrophoresis analysis of mitochondria from different green algal species also indicates that stable dimeric mitochondrial ATP synthases may be characteristic of all Chlorophyceae. One additional subunit, ASA9, was identified in the purified mitochondrial ATP synthase of Polytomella sp. The dissociation profile of the Polytomella enzyme at high-temperatures and cross-linking experiments finally suggest that some of the ASA polypeptides constitute a stator-stalk with a unique architecture, while others may be involved in the formation of a highly-stable dimeric complex. The algal enzyme seems to have modified the structural features of its surrounding scaffold, while conserving almost intact the structure of its catalytic subunits. [less ▲]

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