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J. Biol. Chem., Vol. 283, Issue 38, 26198-26207, September 19, 2008

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The Nucleoside Diphosphate Kinase D (NM23-H4) Binds the Inner Mitochondrial Membrane with High Affinity to Cardiolipin and Couples Nucleotide Transfer with Respiration^*

Malgorzata Tokarska-Schlattner, Mathieu Boissan, Annie Munier, Caroline Borot^¶, Christiane Mailleau, Oliver Speer^||, Uwe Schlattner^||1, and Marie-Lise Lacombe²

From the Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM U884, Université Joseph Fourier, 38041 Grenoble, France, INSERM UMRS_893, CdR Saint-Antoine, UMPC Université Paris 06, 75012 Paris, France, ^¶Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, EA3106, Université Denis Diderot Paris 07, 75005 Paris, France, and ^||ETH Zurich, Institute of Cell Biology, 8093 Zurich, Switzerland

Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on its membrane-bound state.

Received for publication, April 23, 2008 , and in revised form, July 9, 2008.

^* This work was supported by the Germaine de Stael Program for Franco-Swiss collaboration (to U. S. and M.-L. L.), the Agence Nationale de la Recherche (Chaire d'Excellence (to U. S.)), the Marie Curie Intraeuropean Fellowship of the European Community (to M. T.-S.), INSERM, and grants from the Groupement des Entreprises Françaises contre le Cancer and from the Association pour la Recherche contre le Cancer (to M.-L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Université Joseph Fourier, BP 53, 38041 Grenoble, France. Tel.: 33-476-514671; Fax: 33-476-514218; E-mail: uwe.schlattner{at}ujf-grenoble.fr.

² To whom correspondence may be addressed: 27 rue Chaligny, 75012 Paris, France. Tel.: 33-1-4001-1355; Fax: 33-1-4001-1352; E-mail: Marie-Lise.Lacombe{at}inserm.fr.

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