title: The nucleoside diphosphate kinase D (NM23-H4) binds the inner mitochondrial membrane with high affinity to cardiolipin and couples nucleotide transfer with respiration.
abstract: 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.
authors.affiliation: Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM U884, Université Joseph Fourier, 38041 Grenoble, France.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Tokarska-Schlattner
authors.forename: Mathieu
authors.initials: M
authors.lastname: Boissan
authors.forename: Annie
authors.initials: A
authors.lastname: Munier
authors.forename: Caroline
authors.initials: C
authors.lastname: Borot
authors.forename: Christiane
authors.initials: C
authors.lastname: Mailleau
authors.forename: Oliver
authors.initials: O
authors.lastname: Speer
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: Marie-Lise
authors.initials: ML
authors.lastname: Lacombe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002308:Cardiolipins / Q000737:chemistry*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010101:Oxygen Consumption; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
chemical_list: D002308:Cardiolipins; D054778:NM23 Nucleoside Diphosphate Kinases; C517938:NME1 protein, human