Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta.
We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.
Anderca
Marinela Ioana
MI
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan.
Furuichi
Takuya
T
Pinontoan
Reinhard
R
Muto
Shoshi
S
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D000460:Chlorophyta / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D020418:Algal Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009701:Nucleoside-Diphosphate Kinase