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 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

Estructura y función de la ATP sintasa de las arqueas aeróbicas

Ever since Archaea were discovered, their ability to thrive in extreme environments has attracted much attention. Over the years, archaea have gone from microbial extremophilic oddities to organisms of universal importance and have been used to elucidate fundamental biological questions. The phylogeny of the Archaea domain is in constant evolution; to this day it is composed by five main branches: Crenarchaeota, Euryarchaeota, Thaumarchaeota, Korarchaeota and Nanoarchaeota. In the present study, we list the main structural features of the respiratory complexes of the most studied genera of aerobic archaea. We present a morphological comparison of the ATP synthase of these organisms with the rest of the family of rotary ATPases (F- and V-ATPases) as well as a topological analysis of this enzymatic complex (A1Ao -ATP synthase) based on the function of each of the subunits that comprise it

Estructura y función de la ATP sintasa de las arqueas aeróbicas

Ever since Archaea were discovered, their ability to thrive in extreme environments has attracted much attention. Over the years, archaea have gone from microbial extremophilic oddities to organisms of universal importance and have been used to elucidate fundamental biological questions. The phylogeny of the Archaea domain is in constant evolution; to this day it is composed by five main branches: Crenarchaeota, Euryarchaeota, Thaumarchaeota, Korarchaeota and Nanoarchaeota. In the present study, we list the main structural features of the respiratory complexes of the most studied genera of aerobic archaea. We present a morphological comparison of the ATP synthase of these organisms with the rest of the family of rotary ATPases (F- and V-ATPases) as well as a topological analysis of this enzymatic complex (A1Ao-ATP synthase) based on the function of each of the subunits that comprise it.Desde el descubrimiento de las arqueas ha llamado la atención su capacidad para sobrevivir en ambientes difíciles. A través de los años, las arqueas han pasado de ser rarezas extremófilas a ser consideradas organismos de importancia universal que han sido utilizados para elucidar preguntas biológicas fundamentales. La filogenia del dominio Arquea se encuentra en constante cambio y cuenta hasta la fecha con 5 ramas principales: Crenarchaeota, Euryarchaeota, Thaumarchaeota, Korarchaeota y Nanoarchaeota. En el presente trabajo se enlistan las principales características estructurales de los complejos respiratorios de los géneros de arqueas aeróbicas más estudiados. Se presenta una comparación morfológica de la ATP sintasa de estos organismos con el resto de la familia de las ATPasas rotatorias (F- y V-ATPasas); así como un análisis topológico de este complejo enzimático (A1Ao-ATP sintasa) tomando como base la función de cada una de las subunidades que lo conforman

Beyond being an energy supplier, ATP synthase is a sculptor of mitochondrial cristae.

peer reviewedMitochondrial F1FO-ATP synthase plays a key role in cellular bioenergetics; this enzyme is present in all eukaryotic linages except in amitochondriate organisms. Despite its ancestral origin, traceable to the alpha proteobacterial endosymbiotic event, the actual structural diversity of these complexes, due to large differences in their polypeptide composition, reflects an important evolutionary divergence between eukaryotic lineages. We discuss the effect of these structural differences on the oligomerization of the complex and the shape of mitochondrial cristae

Aislamiento de algunas subunidades que integran el brazo periférico de la ATP sintasa mitocondrial de Polytomella sp. y estudio de sus interacciones.

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