The nucleoside diphosphate kinase D (NM23-H4) binds the inner mitochondrial membrane with high affinity to cardiolipin and couples nucleotide transfer with respiration

J Biol Chem. 2008 Sep 19;283(38):26198-207. doi: 10.1074/jbc.M803132200. Epub 2008 Jul 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cardiolipins / chemistry*
  • Humans
  • Intracellular Membranes / metabolism*
  • Male
  • Mitochondria / metabolism*
  • Mitochondria, Liver / metabolism
  • Molecular Sequence Data
  • NM23 Nucleoside Diphosphate Kinases / chemistry
  • NM23 Nucleoside Diphosphate Kinases / genetics
  • NM23 Nucleoside Diphosphate Kinases / physiology*
  • Oxygen Consumption
  • Rats
  • Rats, Wistar
  • Sequence Homology, Amino Acid

Substances

  • Cardiolipins
  • NM23 Nucleoside Diphosphate Kinases
  • NME1 protein, human