1 2 ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro 3 Maya Allouche a , Claire Pertuiset b,c , Jean-Luc Robert d , Cécile Martel b,c , Rémi Veneziano a , Céline Henry e , 4 Ossama Sharaf el dein b,c , Nathalie Saint f , Catherine Brenner b,c,⇑ , Joel Chopineau a,d,⇑ Q1 5 a UMR 5253 CNRS-ENSCM UM2-UM1, Montpellier, France Q2 6 b INSERM UMR-S 769, LabEx LERMIT, Châtenay-Malabry, France 7 c Université Paris-Sud, Châtenay-Malabry, France 8 d Université de Nîmes, Nîmes, France 9 e Plateforme Protéomique, Unité Micalis, INRA Jouy-en-Josas, France 10 f INSERM U1046, Montpellier, France 11 12 14 article info 15 Article history: 16 Received 12 October 2012 17 Available online xxxx 18 Keywords: 19 Channel 20 Liposome 21 Mitochondrion 22 Surface plasmon resonance 23 24 abstract 25 The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central 26 roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between 27 VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in 28 isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and 29 reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 30 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched- 31 preparations of H ANT (ANT from heart, mainly ANT1) and L ANT (ANT from liver, mainly ANT2) isoforms 32 interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and 33 bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform 34 identity and on the conformation of ANT. 35 Ó 2012 Published by Elsevier Inc. 36 37 38 1. Introduction 39 Mitochondria play multiple roles in cellular pathophysiology 40 such as energy and lipid metabolism, reactive oxygen species pro- 41 duction and detoxification and cell death [1]. These organelles are 42 delimitated by two membranes that differ largely by their compo- 43 sition, surface and function. The outer membrane (OM) constitutes 44 a permeable barrier that allows metabolites, ions and water fluxes 45 trough dedicated channels such as the voltage-dependent anion 46 channel or porin (VDAC) [2]. VDAC is the most abundant OM inte- 47 gral protein. It is expressed as three isoforms in mammals, VDAC1 48 to 3, which span the OM with 19 b strands [3]. VDAC channel can 49 be modulated by calcium (Ca 2+ ), anion superoxide, phosphoryla- 50 tion and interaction with cytosolic proteins, all of which influenc- 51 ing cell metabolism and cell fate [4]. In contrast, the inner 52 membrane (IM) delimitates the boundary between the intermem- 53 brane space and the matrix. The exchange of metabolites and ions 54 on IM is mainly mediated by mitochondrial carriers [5]. One of 55 them is the adenine nucleotide translocase (ANT) [6], which is 56 encoded by three or four tissue-specific isoforms in mammals, 57 namely ANT1 to ANT4. In normal conditions, ANT is a stoichiome- 58 tric ADP/ATP translocase, but in the presence of various ligands 59 in vitro (e.g. Ca 2+ ), ANT can behave as a channel, whose conduc- 60 tance can be enhanced by interaction with Bax [7–9]. The bovine 61 ANT1 has been co-crystallized with carboxyatractyloside confirm- 62 ing in silico prediction of six transmembrane helices with N and C 63 terminus facing the intermembrane space [10]. However, despite 64 a lack of consensus on the monomeric versus dimeric status of 65 ANT, it can be found in hetero-oligomers, such as the permeability 66 transition pore (PTP) [8,11,12] and the ATP synthasome [13]. These 67 associations would depend on tissue and pathophysiological con- 68 ditions [14]. 69 Previously, an ANT-VDAC interaction has been evidenced by 70 co-immunoprecipitation and co-localization approaches [11,15]. 71 This interaction might occur at definite contact sites between OM 72 and IM to facilitate metabolites channeling and/or, upon stimula- 73 tion, to trigger a sudden phenomenon named permeability transi- 74 tion (PT) [16]. How ANT and VDAC interact is crucial either for the 75 understanding of basic molecular mechanisms of mitochondrial 76 (dys) function or for drug design, but to our knowledge, the inter- 77 action is poorly characterized. Therefore, we demonstrated for the 78 first time the direct interaction between ANT and VDAC in vitro and 79 unraveled some aspects of its regulation in isolated mitochondria 80 and in a biomimetic system by SPR. 0006-291X/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.bbrc.2012.10.108 ⇑ Corresponding authors. Address: INSERM UMR-S 769, LabEx LERMIT, Châtenay- Malabry, France (C. Brenner), UMR 5253 CNRS-ENSCM UM2-UM1, Montpellier, France (J. Chopineau). E-mail addresses: catherine.brenner-jan@u-psud.fr (C. Brenner), joel.chopineau @unimes.fr (J. Chopineau). Biochemical and Biophysical Research Communications xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 29481 No. of Pages 6, Model 5G 9 November 2012 Please cite this article in press as: M. Allouche et al., ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro, Biochem. Biophys. Res. Commun. (2012), http://dx.doi.org/10.1016/j.bbrc.2012.10.108