[Frontiers in Bioscience E3, 395-409, January 1, 2011]

The respiratory-dependent assembly of ANT1 regulates cytochrome c release

Faustin Benjamin 1, Rossignol Rodrigue 1, Deniaud Aurelien 2, Rocher Christophe 1, Claverol Stephane 3, Malgat Monique1, Brenner Catherine 4, Letellier Thierry 1

1INSERM U688, Universite Victor Segalen-Bordeaux 2, 146 rue Leo-Saignat, F-33076 Bordeaux Cedex, France, 2University of Versailles/St Quentin, PRES UniverSud Paris, CNRS UMR 8159, Bat Buffon, 45 avenue des Etats-Unis, F-78035 Versailles, France, 3Centre de genomique fonctionnelle, Universite Victor Segalen-Bordeaux 2, 146 rue Leo-Saignat, F-33076 Bordeaux Cedex, France, 4 Univ Paris-Sud, INSERM UMR-S 769, PRES UniverSud Paris, Chatenay-Malabry, 92290, France

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
3.1. Animals and mitochondrial preparations
3.2. Human muscle biopsies
3.3. Oxygraphic and swelling measurements
3.4. Titration curves, threshold curves, and threshold value determination
3.5. Production and purification of recombinant BaxDC
3.6. Electrophoretic techniques
3.7. Western-blot
3.8. Protein detection and analysis
4. Results
4.1. Distribution of ANT1 oligomeric and conformational states in functional mitochondria
4.2. Oligomeric and conformational states of ANT1 depend on respiratory chain activity
4.3. ANT may be mobilized from a structure responsible for the mitochondrial permeability transition
4.4. Cytochrome c is mobilized through the respiratory-induced oligomerization of ANT1
4.5. Respiratory-induced structural changes in ANT1 regulate Bax-triggered cytochrome c release from mitochondria
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

The adenine nucleotide translocator (ANT) is a control point of several fundamental cell processes, as diverse as cell energy supply, mitochondrial DNA maintenance, and apoptosis. This paper describes six individual structures of the carrier, distinguished according to ANT1 oligomeric and conformational states, as well as associations with other proteins. Transitions between these structures depend on energy demand and thus contribute to a metabolic reserve of oxidative phosphorylation (OXPHOS) activity. Moreover, at low respiratory chain activity, we demonstrate that, unlike a mitochondrial Ca2+ upload, Bax, a pro-apoptotic Bcl-2-family protein, is able to trigger a massive release of cytochrome c from one of these ANT1 structures. These new insights emphasize the close relationship between structural rearrangements of ANT and molecular apoptotic events at distinct cell energy levels. OXPHOS functioning has to therefore be considered a crucial control point for the events leading to these contrasting pathways.