Figure 8.

Expression of METTL1[S27A], but not S27D or S27E, complements a yeast trm8-Δ mutant. (A) Complementation of yeast trm8 mutants by METTL1 and Ser27 variants. A trm8-Δ yeast strain AA0636 (Alexandrov et al, 2005), containing a trm4 mutation to enhance its phenotype (see Materials and methods), was transformed with plasmids expressing galactose-inducible (P_GAL controlled) WDR4, METTL1, METTL1 variants, TRM8 or vector controls as indicated (Alexandrov et al, 2002), and tested for growth by serial 10-fold dilution of cultures (starting from 4 × 10³ cells), followed by incubation at the indicated temperatures on synthetic media containing galactose or glucose, as described in Materials and methods. (B) Analysis of expression of METTL1 and Ser27 variants. Strains described in panel A were grown as described, harvested after 5 h induction in media containing galactose, and crude lysates analysed by immunoblotting using the antibody recognising both phosphorylated and unphosphorylated forms of METTL1. Lanes a–d and f contain the METTL1 variants indicated.