Journal Article

Identification of a Mitochondrial Nucleoside Diphosphate Kinase from the Green Alga <i>Dunaliella tertiolecta</i>

Marinela Ioana Anderca, Takuya Furuichi, Reinhard Pinontoan and Shoshi Muto

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 11, pages 1276-1284
Published in print November 2002 | ISSN: 0032-0781
Published online November 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf155
Identification of a Mitochondrial Nucleoside Diphosphate Kinase from the Green Alga Dunaliella tertiolecta

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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.

Keywords: Keywords: cDNA cloning — Dunaliella tertiolecta — Green fluorescent protein — Mitochondrial isoform — Nucleoside diphosphate kinase — Sequence analysis.; Abbreviations: BSA, bovine serum albumin; GFP, green fluorescent protein; NDP kinase, nucleoside diphosphate kinase (EC 2.7.4.6); RACE-PCR, rapid amplification of cDNA ends–PCR;

Journal Article.  5890 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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