The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase.
We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.
Milon
L
L
INSERM U402, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75012 Paris, France.
Meyer
P
P
Chiadmi
M
M
Munier
A
A
Johansson
M
M
Karlsson
A
A
Lascu
I
I
Capeau
J
J
Janin
J
J
Lacombe
M L
ML
D001483:Base Sequence; D002461:Cell Line, Transformed; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D054778:NM23 Nucleoside Diphosphate Kinases; D061107:Nucleoside Diphosphate Kinase D; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; C517938:NME1 protein, human; C517946:NME4 protein, human; D061107:Nucleoside Diphosphate Kinase D; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins