Complementation of Trl1 defects by orthologs from fungal pathogens. (A) The Trl1 orthologs of representative pathogenic fungi complement trl1Δ, except the sealing-only Trl1s of Mucorales. The indicated Trl1 orthologs were expressed in S. cerevisiae trl1Δ [TRL1, URA3] strain background and subjected to serial dilution growth assay on media containing 5-FOA (selecting against the TRL1 plasmid) or control media. (B–D) The Mucorales Trl1 can complement a defect in the ligase domain of ScTrl1 (B) but is unable to complement the defects in the kinase (C) or CPD (D) domain of ScTrl1. The indicated Trl1 orthologs were expressed in a strain that contains a URA3 plasmid with a wild-type TRL1 gene and another plasmid with either the sealing-defective trl1-K114A (B), the kinase-defective trl1-K404AT405A (C), or the CPD-defective trl1-H777A (D) subjected to serial dilution growth assay on media containing 5-FOA (selecting against the TRL1 plasmid) or control media. (E) A conserved lysine in the active site is critical in the Mucorales Trl1s. Amino acid changes orthologous to ScTrl1-K114A were introduced in Mucorales Trl1 and subjected to plasmid shuffle assay in the sealing-defective ScTrl1 strain as in B.
