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Abstract
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.
Highlights
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
Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity
The present work demonstrates that nm23-H4, a recently discovered member of the nm23-NDP kinase gene family, encodes a catalytically active NDP kinase that is localized in mitochondria
Summary
Materials—Transformed human embryonic kidney (HEK) 293 cells were obtained from the ATCC. The eluted fractions containing the recombinant protein were equilibrated in 50 mM HEPES, pH 8, buffer containing 5 mM dithiothreitol and 5 mM EDTA by chromatography on a PD10 column (Amersham Pharmacia Biotech). To assay the NDP kinase activity of the full-length Nm23-H4 after renaturation, the protein eluted from the nickel-nitrilotriacetic acid column in urea containing buffer (Qiagen) was incubated overnight at room temperature after a 100-fold dilution in the NDP kinase reaction buffer devoid of pyruvate kinase, lactate dehydrogenase, and dTDP. To determine the stability upon urea treatment, the enzymes were diluted at the same concentration as above in 50 mM HEPES, pH 8, 10% glycerol and 0.1 M NaCl containing the indicated concentrations of urea and were incubated for 16 h at room temperature Residual activity after both heat and urea treatments was measured by the coupled assay after a 100-fold dilution in the reaction buffer.
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