Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

Abstract

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6xHis-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.