Mitochondrial Physiology Network 10.9: 48 (2005) - download pdf

Mitochondrial GTP metabolism - a main function of NDP-kinase D?

Uwe Schlattner¹, M Tokarska-Schlattner¹, M Boissan², O Speer¹, A Munier²,

M-L Lacombe²

¹Swiss Federal Inst. Technology (ETH), Inst. Cell Biology, Zurich, Switzerland; ²INSERM U402, Fac. Médecine Saint-Antoine, 75012 Paris, France. - uwe.schlattner@cell.biol.ethz.ch

    Isoenzymes of hexameric nucleoside diphosphate kinase (NDPK/nm23) function in NTP-bio­synthesis (NDP + ATP <-> NTP + ADP) and have additional, multi-facetted roles in cell signaling, proliferation and differentiation [1]. Detailed subfractionation of rat liver cells and surface plasmon resonance spectroscopy [2] with recombinant protein revealed that the NDPK-D isoenzyme [3] has a unique intramitochondrial localization at the inner mitochondrial membrane, where it firmly binds to acidic phospholipids, mainly cardiolipin. The latter high affinity interaction (K_D about 30 nM) is due to the NDPK-D-specific arginine 90 in a basic RRK motif at the surface of the NDPK hexamer, since a R90D mutation abolishes cardiolipin interaction. Latency assays with liver, HEK and HeLa mitochondria suggest that most NDPK-D is oriented towards the intermembrane and cristae space, while a variable fraction may be oriented towards the matrix space. The physiological role of NDPK-D was analyzed with HeLa cell lines that can express NDPK-D under the control of an inducible tetracycline (tet) promoter. Mitochondrial respiration from control cells was only weakly stimulated with NDPK substrate TDP, while it was strongly stimulated in NDPK-D overexpressing  tet-treated cells, together with a decrease in K_m (ADP). Thus, NDPK-D uses NDP nucleotides to locally regenerate ADP in the mitochondrial intermembrane space, which in turn stimulates oxidative phosphorylation. From the NTP generated, mainly GTP could be important for GTP-dependent processes in the intermembrane compartment, like e.g. GTP-binding proteins involved in mitochondrial dynamics.

   We propose a model for NDPK-D participating in GTP-export from the matrix space and GTP-regeneration in the intermembrane space by association of the NDP kinase hexamer with adenylate translocator of the inner membrane via cardiolipin patches, similar to proteolipid complexes formed by octameric mitochondrial creatine kinase [4].

    Supported by the Germaine de Stael Program for Swiss-French collaboration.

1.  Lacombe ML, Milon L, Munier A, Mehus JG, Lambeth DO (2000) The human Nm23/nucleoside diphosphate kinases. J. Bioenerg. Biomembr. 32: 247-258.

2.  Schlattner U, Wallimann T (2000) A quantitative approach to membrane binding of human ubiquitous creatine kinase. J. Bioenerg. Biomembr. 32: 123-131.

3.  Milon L, Meyer P, Chiadmi M, Munier A, Johansson M, Karlsson A, Lascu I, Capeau J, Janin J, Lacombe ML (2000) The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase. J. Biol. Chem. 275: 14264-14272.

4   Schlattner U, Gehring F, Vernoux N, Tokarska-Schlattner M, Neumann D, Marcillat O, Vial C, Wallimann T (2004) C-terminal lysines determine phospholipid interaction of mitochondrial creatine kinase. J. Biol. Chem. 279: 24334-24342.