TEMPORAL AND SPATIAL DISTRIBUTIONS OF YEAST NUCLEOSIDE DIPHOSPHATE KINASE-ACTIVITIES AND ITS ASSOCIATION WITH THE CDC8P

Nucleoside diphosphate kinase (E.C. 2.7.4.6.) is a broad substrate-spe cific enzyme that catalyzes the phosphorylation of nucleoside diphosph ates to the corresponding triphosphates in nucleic acid biosynthesis. In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s). Our results show that the enzyme is predominantly cytoplasmic. A substant ial amount of enzyme activity (40-50%) may be associated with the cell membrane. Less than 1% of total activity was detected in the nuclear fraction. Approximately 3% was found in the mitochondrial fraction. Wh en yeast cultures were synchronized, we found that Saccharomyces cerev isiae nucleoside diphosphate kinase did not show cell cycle periodicit y, as Schizosaccharomyces pombe enzyme did. To explore its link with D NA synthesis, we investigated its relationship with the Cdc8p (dTMP ki nase). We demonstrated a physical interaction between these proteins i n vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract. Furthermore, when GST:Cdc8p protein was expresse d in yeast, the protein could bind to the glutathione-agarose, along w ith nucleoside diphosphate kinase, suggesting that there is an interac tion between GST:Cdc8p and nucleoside diphosphate kinase in vivo. Our results provide evidence for at Least a two-enzyme complex that may we ll facilitate nucleotide channeling in the cell.