ISOLATION AND CHARACTERIZATION OF THE CANDIDA-ALBICANS GENE FOR MESSENGER-RNA 5'-TRIPHOSPHATASE - ASSOCIATION OF MESSENGER-RNA 5'-TRIPHOSPHATASE AND MESSENGER-RNA 5'-GUANYLYLTRANSFERASE ACTIVITIES IS ESSENTIALFOR THE FUNCTION OF MESSENGER-RNA 5'-CAPPING ENZYME IN-VIVO

The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-tripho sphatase (TPase) diverges from those of higher eukaryotes, in order to confirm the sequence divergence of TPases in lower and higher eukaryo tes, the Candida albicans gene for TPase was identified and characteri zed. This gene designated CaCET1 ((<(C.)under bar>) (a) under bar lbic ans mRNA 5'-(c) under bar apping enzyme triphosphatase (1) under bar) has an open reading frame of 1.5 kb, which can encode a 59-kDa protein . Although the N-terminal one-fifth of S. cerevisiae TPase (ScCet1p) i s missing in CaCet1p, CaCet1p shares significant sequence similarity w ith ScCet1p over the entire region of the protein; the recombinant CaC et1p, which was expressed as a fusion protein with glutathione S-trans ferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-d eficient S. cerevisiae cells when expressed under the control of the A DH1 promoter, whereas the human capping enzyme derivatives that are ac tive for TPase activity but defective in mRNA 5'-guanylyltransferase ( GTase) activity did not. Yeast two-hybrid analysis revealed that C. al bicans Cet1p fan bind to the S. cerevisiae GTase in addition to its ow n partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast G Tase. These results indicate that the failure of the human TPase activ ity to complement an S. cerevisiae cet1 Delta null mutation is attribu table, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association o f GTase and TPase is essential for the function of the capping enzyme in vivo. (C) 1998 Federation of European Biochemical Societies.