Subunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

peer reviewedMitochondrial 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

Dissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.

peer reviewedThe algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

AbstractTwo genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart

The Peripheral Stalk of Rotary ATPases

Rotary ATPases are a family of enzymes that are thought of as molecular nanomotors and are classified in three types: F, A, and V-type ATPases. Two members (F and A-type) can synthesize and hydrolyze ATP, depending on the energetic needs of the cell, while the V-type enzyme exhibits only a hydrolytic activity. The overall architecture of all these enzymes is conserved and three main sectors are distinguished: a catalytic core, a rotor and a stator or peripheral stalk. The peripheral stalks of the A and V-types are highly conserved in both structure and function, however, the F-type peripheral stalks have divergent structures. Furthermore, the peripheral stalk has other roles beyond its stator function, as evidenced by several biochemical and recent structural studies. This review describes the information regarding the organization of the peripheral stalk components of F, A, and V-ATPases, highlighting the key differences between the studied enzymes, as well as the different processes in which the structure is involved

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

De homologías y embarazos: cómo se perpetúa un error conceptual en la literatura científica

One of the most abused terms in biological scientific literature is the word ‘homology’, whose erroneous use is widely spread. We often find phrases that contain the terms ‘percentage of homology’ or ‘highly homologous’, or even ‘low homology’, which seem to give the word ‘homology’ a quantitative value, to which a numerical value can be assignedUno de los términos más maltratados en la literatura científica biológica es la palabra «homología», cuya utilización errónea está ampliamente extendida. Con frecuencia encontramos frases que contienen los términos «porcentaje de homología» o «altamente homólogo», o bien «baja homología», que parecen otorgar a la palabra «homología» un valor cuantitativo, al que puede asignársele un valor numéric

Topología y función de las subunidades intrínsecas de la membrana de las F1FO-ATP sintasa mitocondriales

La F1FO-ATP sintasa es un complejo enzimático que se encuentra ampliamente distribuido en las membranas transductoras de energía. Todas las ATPasas, incluidas las mitocondriales, cloroplastídicas y bacterianas, comparten similitudes estructurales y funcionales. Sin embargo, hay diferencias en su composición quedependen de la especie, siendo más compleja en organismos como Saccharomyces cerevisiae o Bostaurus. Es por ello que una mejor comprensión de la estructura de la F1FO-ATP sintasa contribuirá a un mayorconocimiento a nivel molecular tanto de la función como de la regulación de este complejo enzimático. En la actualidad, se sabe muy poco acerca de la organización estructural de las subunidades de la región FO. Considerando lo anterior, en este trabajo se presenta información concerniente a las proteínas intrínsecas del dominio FO de las ATP sintasas más investigadas a la fecha, así como de algunas otras subunidades de membrana que se encuentran presentes en organismos menos estudiados

Topología y función de las subunidades intrínsecas de la membrana de las f1fo-ATP sintasa mitocondriales

La F 1 F O -ATP sintasa es un complejo enzimático que se encuentra ampliamente distribuido en las membranas transductoras de energía. Todas las ATPasas, incluidas las mitocondriales, cloroplastídicas y bacterianas, comparten similitudes estructurales y funcionales. Sin embargo, hay diferencias en su composición que dependen de la especie, siendo más compleja en organismos como Saccharomyces cerevisiae o Bos taurus . Es por ello que una mejor comprensión de la estructura de la F 1 F O -ATP sintasa contribuirá a un mayor conocimiento a nivel molecular tanto de la función como de la regulación de este complejo enzimático. En la actualidad, se sabe muy poco acerca de la organización estructural de las subunidades de la región F O. Considerando lo anterior, en este trabajo se presenta información concerniente a las proteínas intrínsecas del dominio F O de las ATP sintasas más investigadas a la fecha, así como de algunas otras subunidades de membrana que se encuentran presentes en organismos menos estudiados

Topología y función de las subunidades intrínsecas de la membrana de las F1FO-ATP sintasa mitocondriales

The F1FO-ATP synthase is a complex widely distributed in energy-transducing membranes. All ATPases, including the mitochondrial, chloroplastic and bacterial, share structural and functional similarities. However, there are differences in their composition that depend on the species, being more complex in organisms such as Saccharomyces cerevisiae or Bos taurus. This is why, a better understanding of the F1FO-ATP synthase structure will contribute to a greater knowledge at a molecular level, both of the function, and the regulation of this enzymatic complex. At present, very little is known about the structural organization of the subunits from the FO domain. Considering the former, this paper presents information concerning the intrinsic membrane proteins from the most researched F1FO-ATP synthases to date, as well as some other membrane subunits present in less studied organisms. La F1FO-ATP sintasa es un complejo enzimático que se encuentra ampliamente distribuido en las membranas transductoras de energía. Todas las ATPasas, incluidas las mitocondriales, cloroplastídicas y bacterianas, comparten similitudes estructurales y funcionales. Sin embargo, hay diferencias en su composición que dependen de la especie, siendo más compleja en organismos como Saccharomyces cerevisiae o Bos taurus. Es por ello que una mejor comprensión de la estructura de la F1FO-ATP sintasa contribuirá a un mayor conocimiento a nivel molecular tanto de la función como de la regulación de este complejo enzimático. En la actualidad, se sabe muy poco acerca de la organización estructural de las subunidades de la región FO. Considerando lo anterior, en este trabajo se presenta información concerniente a las proteínas intrínsecas del dominio FO de las ATP sintasas más investigadas a la fecha, así como de algunas otras subunidades de membrana que se encuentran presentes en organismos menos estudiados

La expresión alotópica: ¿tarea imposible o estrategia factible para el tratamiento de enfermedades mitocondriales?

Allotopic expression refers to the functional relocation of genes from one cellular compartment to another. Here, it refers to the expression of mitochondrial genes either from the nucleus or from a cytosolic vector. It is considered a promising strategy to develop gene therapies against diseases caused by mutations in the mitochondrial genome. Nowadays, it is possible to introduce genetic material into the nuclear chromosomes and there is a good knowledge about the mechanisms of protein import into mitochondria so in principle, a single gene copy per cell would be sufficient to synthesize the necessary proteins and deliver them to all affected mitochondria. In the last 20 years allotopic expression has been tried in yeasts, plants, mammal cells and animals carrying mitochondrial diseases. Many studies published in the last 10 years suggest that allotopic expression is a successful strategy and some clinical trials using this approach are currently being carried out. Nevertheless, there is also a large number of evidence that questions the viability of allotopic expression and that proposes more stringent criteria to make sure that the allotopically-expressed proteins are actually assembled into their corresponding mitochondrial complex. In this work, we first introduce the concept of allotopic expression, then we review the most relevant data in the field and, finally, we discuss the difficulties that must be overcome before attempting gene therapies in patients with mitochondrial diseases.La expresión alotópica es la relocalización funcional de genes de un compartimento celular a otro. En este trabajo, se refiere a la expresión de genes mitocondriales desde el núcleo o desde un vector citosólico. Se considera que esta estrategia es una de las más prometedoras para desarrollar terapias génicas para el eventual tratamiento de enfermedades ocasionadas por mutaciones en el genoma mitocondrial. Actualmente, existen herramientas para introducir material genético en los cromosomas nucleares, se tiene un conocimiento razonable de los mecanismos de importación de proteínas a la mitocondria y en teoría, una sola copia del gen por célula es suficiente para que se sintetice la proteína necesaria y se distribuya en todas las mitocondrias afectadas. Durante los últimos 20 años se ha intentado la expresión alotópica de diversos genes mitocondriales en levaduras, plantas y células de mamífero, así como en animales con enfermedades mitocondriales. Muchos trabajos publicados en los últimos 10 años apuntaban a que la expresión alotópica era una estrategia exitosa, al grado de que han autorizado los primeros estudios clínicos para tratar mediante terapia génica a pacientes con enfermedades mitocondriales. Sin embargo, también se han reportado evidencias que cuestionan la viabilidad de la expresión alotópica y que proponen nuevos criterios para asegurar que las proteínas expresadas alotópicamente verdaderamente se integraron al complejo mitocondrial al cual pertenecen. En este trabajo, presentamos una breve introducción de lo que es la expresión alotópica, hacemos una revisión de los resultados más relevantes en el campo de la expresión de genes mitocondriales desde el núcleo y discutimos las dificultades que consideramos que hay que sortear antes de proponer terapias génicas para combatir con éxito las enfermedades mitocondriales