Contents lists available at ScienceDirect BBA - Bioenergetics journal homepage: www.elsevier.com/locate/bbabio Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga Lorenzo Sánchez-Vásquez a , Miriam Vázquez-Acevedo a , Javier de la Mora a , Félix Vega-deLuna a , Pierre Cardol b , Claire Remacle b , Georges Dreyfus a , Diego González-Halphen a,⁎ a Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, México b Genetics and Physiology of Microalgae, University of Liège, B-4000 Liège, Belgium ARTICLE INFO Keywords: F 1 F O -ATP synthase peripheral arm Membrane domain of the ATP synthase Chlorophycean algae Chlamydomonas reinhardtii Polytomella sp. Asa subunits ABSTRACT Mitochondrial F 1 F O -ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600 kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, ε, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6–Asa6, Asa6–Asa8, Asa6–Asa9, Asa8–Asa8 and Asa8–Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices. 1. Introduction Mitochondrial F 1 F O -ATP synthase (complex V) is a key component of oxidative phosphorylation. The oligomeric membrane complex exhibits two distinct domains: i) the F O sector, comprising a mem- brane-bound, proton-translocating unit formed by subunit a and an oligomer of c subunits, and a dimerization module formed by subunits A6L, e, f and g; and ii) the F 1 extrinsic domain that comprises the catalytic core formed by 3 α and 3 β subunits, a central rotary shaft built by subunits γ, δ and ε, and a peripheral-stalk constructed by subunits OSCP, b, d and F6 (beef heart enzyme nomenclature) [1]. The enzyme is considered to work as a molecular rotary motor with stator and motor components that are driven by the electrochemical proton gradient. Proton translocation occurs through two offset channels shared by subunit a and the ring-shaped c-subunit oligomer. The c-ring rotates along with the central rotor stalk (subunits γ, δ and ε) which in turn rotates relative to the α 3 /β 3 catalytic moiety in 120° stepped increments inducing three conformational states in the nucleotide-binding β- subunits, according to the Brownian ratchet mechanism, leading to ATP synthesis [2,3]. Indeed, this was proven by direct visualization of the rotation of an actin filament attached to the γ subunit of bacterial F 1 -ATPase [4]. In recent years, it has become evident that all mitochondrial ATP synthases exhibit a well-conserved basic core formed by subunits α 3 /β 3 / γ/δ/ε/c/a/OSCP, but that different organisms contain atypical poly- peptides in the peripheral stalk and/or in the dimerization modules. Thus, in ciliates [5], trypanosomatids [6], euglenoids [7], and chlor- ophycean algae [8], a variety of proteins seem to substitute the conventional b, d, F6, A6L, e, f and g subunits (beef heart enzyme nomenclature). The mitochondrial F 1 F O -ATP synthase of chlorophycean algae can be isolated as a stable dimer of 1600 kDa after solubilizing mitochon- dria with detergents such as n-dodecyl-β-D-maltoside [9]. As in all known eukaryotes, the rotary and catalytic cores of the algal enzyme contain the eight conserved subunits mentioned above [10]. Never- theless, nine non-conventional subunits (Asa1 to Asa9), unique to the mitochondrial ATP synthases of chlorophycean algae, are also consti- tuents of the enzyme. Some Asa subunits form the robust peripheral http://dx.doi.org/10.1016/j.bbabio.2017.04.004 Received 24 January 2017; Received in revised form 25 April 2017; Accepted 29 April 2017 ⁎ Corresponding author at: Departamento de Genética Molecular, Instituto de Fisiología Celular, UNAM, Apartado Postal 70-600, Delegación Coyoacán, 04510, México D.F., Mexico. E-mail address: dhalphen@ifc.unam.mx (D. González-Halphen). BBA - Bioenergetics 1858 (2017) 497–509 Available online 01 May 2017 0005-2728/ © 2017 Elsevier B.V. All rights reserved. MARK