The Journal of Experimental Medicine
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Published online 19 February 2001.
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© The Rockefeller University Press, 0022-1007/2001/2/509/ $5.00
The Journal of Experimental Medicine, Volume 193, Number 4, February 19, 2001 509-520

Original Article

Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2

Etienne Jacotota, Karine F. Ferria, Chahrazed El Hamela, Catherine Brennera,c, Sabine Druillennecb, Johan Hoebeked, Pierre Rustine, Didier Métiviera, Christine Lenoirb, Maurice Geuskensf, Helena L.A. Vieiraa, Markus Loefflera, Anne-Sophie Belzacqc, Jean-Paul Briandd, Naoufal Zamzamia, Lena Edelmang, Zhi Hua Xieh, John C. Reedh, Bernard P. Roquesb, and Guido Kroemera

a Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F-94805 Villejuif, France
b Unité de Pharmacochimie Moléculaire et Structurale, Institut National de la Santé et de la Recherche Médicale U266, Centre National de la Recherche Scientifique, UMR 860, Université René Descartes (Paris V), 75006 France
c Centre National de la Recherche Scientifique, UMR A6022, Université de Technologie de Compiègne, F-60205 Compiègne, France
d Institut de Biologie Moleculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, UPR 9021, Centre National de la Recherche Scientifique, 67084 Strasbourg, France
e Unité de Recherche sur les Handicaps Génétiques de l'Enfant (Institut National de la Santé et de la Recherche Médicale U393), Hôpital des Enfants Malades-Necker, 75015 Paris, France
f Free University of Brussels, B-1640 Rhode-St-Genese, Belgium
g Laboratoire de Technologie Cellulaire, Institut Pasteur, 75015 Paris, France
h The Burnham Institute, La Jolla, California 92037
CNRS-UMR 1599, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.33-1-42-11-60-46

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein–protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96–induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT–Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT–Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Key Words: ADP/ATP translocase • HIV • Vpr • mitochondria • Bcl-2

Abbreviations used in this paper: ANT, adenine nucleotide translocator; Atr, atractyloside; BA, bongkrekic acid; CCCP, carbonylcyanide m-chlorophenylhydrazone; CsA, cyclosporin A; {Delta}{Psi}m, mitochondrial transmembrane potential; FSC, forward scatter; IM, inner membrane; MMP, mitochondrial membrane permeabilization; OM, outer membrane; PA10, König's polyanion; PC, phosphatidylcholine; PTPC, permeability transition pore complex; RC, respiratory control; RT, room temperature; VDAC, voltage-dependent anion channel; Vpr, viral protein R.

© 2001 The Rockefeller University Press


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