FIG. 1.

In vivo analysis of Bur1 mutants. (A) Schematic of the Bur1 protein showing the residues targeted for mutation. (B) The mutant proteins are expressed at normal levels. The indicated HA₃-tagged BUR1 alleles were used to replace the wild-type gene by plasmid shuffling. Whole-cell extracts were resolved by SDS-PAGE, and the expression levels were assayed by immunoblotting with anti-HA (12CA5) antibody. The very dark band near 50 kDa is a protein that cross-reacts with the 12CA5 antibody. FL, the full-length protein; CΔ, the truncation at amino acid 393; Vect, the vector plasmid with no BUR1 gene. (C) Plasmid shuffling was performed on synthetic complete (SC) medium containing 5-FOA. All mutants except E107Q and D213A were able to support growth. (D) Cold-sensitive phenotypes of Bur1 T-loop and CTD mutants. Strains from panel C were spotted on various media for further analysis at the indicated temperatures. Growth at 30, 12, and 37°C was on yeast extract-peptone-dextrose (YPD); Caff, SC plus 15 mM caffeine; Form, YPD plus 2% formamide. (E) Spt and Bur phenotypes of mutants. Strains from panel C were assayed for Spt and Bur phenotypes. Spt⁻, suppression of lys2-128δ allowing growth on SC-lysine; Bur⁻, ability to bypass suc2ΔUAS (−1900 to −390) and support growth on 2% sucrose (+1 μg of actinomycin A/ml) as sole carbon source. (F) Overexpression of Bur2 partially suppresses the cold-sensitive (cs) phenotype of the T240A and T240D alleles. Strains were derived by plasmid shuffling and further transformed with high-copy-number vector (pRS424) or the same plasmid carrying the BUR2 gene. Strains were spotted onto YPD and grown at 12 or 30°C.