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

Abstract

The 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 1600 kDa. 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.

Amphipol‐assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii

“…) would be useful for investigating precise excitation energy transfer by means of fluorescence analyses at room temperature. Although APols were invented more than 20 years ago, only a limited number of studies to date have succeeded in stabilizing and characterizing the giant (> 1 MDa) membrane supercomplexes . This APol‐assisted purification method combined with additional stabilizer(s) may also be useful in protein supercomplex biology in other fields.…”

Morphological, physiological, and transcriptional responses of the freshwater diatom Fragilaria crotonensis to elevated pH conditions

“…In contrast, genes classified within "cellular respiration" exhibited an overall increase in relative expression at pH 9.2 (Figure 3B). Representation of genes involved in oxidative phosphorylation increased at pH 9.2, including NADH quinone oxidoreductase chain 5 and 6 genes (ND5) (ND6) (Melo et al, 2004) and ATP synthase subunit α gene (ATP6) (Vázquez-Acevedo et al, 2016). In contrast, mitochondrial transport genes decreased in relative expression at high pH, including carrier gene (SLC25) (Ruprecht and Kunji, 2020).…”

Topología Y Función De Las Subunidades Intrínsecas De La Membrana De Las F 1 F O -Atp Sintasa Mitocondriales

“…Asimismo, Asa8 se compone de 89 aminoácidos (9.9 kDa), presenta un cruce transmembranal y una topología de membrana N-adentro C-fuera, deducido a partir de análisis bioinformáticos. Debido a que la subunidad Asa8 contiene un dominio GxxxG (Gly59-Gly63) podría formar homodímeros y de esta forma participar en la dimerización de la enzima (Vázquez-Acevedo et al, 2016), ya que la presencia de estos dominios en las subunidades e y g de levadura desempeñan un papel importante en la formación de estructuras diméricas del complejo enzimático (Arselin et al, 2003;Arselin et al, 2004;Bustos & Velours, 2005). Por último, Asa9 está formada por 97 aminoácidos y tiene una masa molecular estimada de 11 kDa.…”

“…La enzima de la mayoría de los organismos eucariontes, desde las levaduras hasta las plantas y los mamíferos, cuentan con las mismas subunidades, aunque poco conservadas a nivel de secuencia pero más conservadas a nivel de estructura, lo cual indica que estas subunidades comparten la misma función dentro del complejo (Vázquez-Acevedo et al, 2016).…”