pct1 is an mRNA-capping RTPase but does not associate with pce1. (A) Complementation by plasmid shuffling. pct1 plus the indicated GTases were tested for the ability to replace Ceg1 and Cet1 as described in Materials and Methods. (B) Immunoprecipitation and GTase-GMP formation assay. Whole-cell extracts were prepared from strains derived from S. cerevisiae YSB719 (lanes 1 and 2) or S. pombe TE696 strain (lanes 3 to 6) expressing the indicated proteins. RTPases were immunoprecipitated (immunoblot shown in top panel) and pellets were tested for the presence of GTase (bottom panel), as described in Materials and Methods. The asterisk denotes radioactive phosphate.

Different interaction patterns between the CTD and fungal capping enzyme subunits. CTD peptides consisting of four heptapeptide repeats were conjugated to beads. The peptides were either unphosphorylated (CTD) or phosphorylated at all four serine 5 positions (CTD-P). Beads were incubated with recombinant fungal GTases and RTPases as described in Materials and Methods. Proteins bound to peptides were incubated with [α-³²P]GTP to assay guanylyltransferase activity. Bound proteins were analyzed by SDS-PAGE followed by transfer to nitrocellulose membranes. Blots were analyzed by immunoblotting with anti-His₆ monoclonal antibody (upper panels) and autoradiography (bottom panel). The asterisk denotes the position of the radioactive phosphate.

The interaction between Cgt1 and CaCet1 is not absolutely required for their function in vivo. (A) Sequence alignment between CaCet1 (GenBank accession number O93813, residues 193 to 292) and Cet1 (O13297, residues 232 to 310). Protein sequence similarity searching was carried out on the National Center for Biotechnology Information Web server using the BLAST algorithm (1), and sequence alignments were made using SEQVU. Letters represent the single-letter amino acid code, and numbers represent the positions of the amino acid residues. Boxed residues denote identities, and shaded residues indicate similar amino acids. Asterisks indicate the residues that are important for the Cet1-Ceg1 interaction (48). The residues used for the deletion of Cet1 and CaCet1 are shown. (B) Deletion analysis of CaCet1. The indicated deletions of CaCet1 were tested for the ability to replace Cet1 (see Materials and Methods) in the presence of Ceg1 or Cgt1. Plates are shown after 3 days at 30°C. (C) Immunoprecipitation and GTase-GMP formation assay for CaCet1 derivatives and Cgt1, respectively (see Materials and Methods). Upper panel, immunoblotting with 12CA5; and lower panel, autoradiography. (D) Coexpression of Cet1 mutants and various GTases in S. cerevisiae. Plasmid shuffling with the indicated genes was carried out as described in Materials and Methods. After 2 days, 5-FOA-resistant cells were spotted on new plates and were further incubated either at 30 or 37°C. + indicates that the cells form colonies after 3 days. − indicates that colonies were not observed after 7 days. Note that the mouse capping enzyme results are from Takase et al. (48).