Isolation and characterization of the Candida albicans gene for mRNA 5'-triphosphatase: association of mRNA 5'-triphosphatase and mRNA 5'-guanylyltransferase activities is essential for the function of mRNA 5'-capping enzyme in vivo.
The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C. albicans mRNA 5'-capping enzyme triphosphatase 1) 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) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S-transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5'-guanylyltransferase (GTase) activity did not. Yeast two-hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1delta null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.
Yamada-Okabe
T
T
Department of Hygiene, Yokohama City University, Japan.
Mio
T
T
Matsui
M
M
Kashima
Y
Y
Arisawa
M
M
Yamada-Okabe
H
H
D017766:Acid Anhydride Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D000098556:mRNA Guanylyltransferases
D005656:Fungal Proteins; D012315:RNA Caps; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase