Figure 4.

Growth complementation of an Mtr4-knockout strain by ratchet helix mutants. (A) Slow growth phenotypes are observed for each of the Mtr4 ratchet helix mutants. The indicated plasmids were introduced into a yeast strain that had the Mtr4^WT gene deleted from the chromosome, and that also contained a plasmid encoding Mtr4^WT with an URA3 selectable marker. Growth on 5-FOA plates selects for cells that have lost the URA3 plasmid, and thus shows a slow growth phenotype associated with each mutant. Mtr4^archless has a more severe slow growth phenotype than any of the ratchet helix mutants. Mtr4^D262A/E263A removes residues required for the binding and hydrolysis of ATP and is used as a negative control. (B) Double mutants of ratchet helix point mutations combined with an arch deletion. No growth is observed for the Mtr4^{R1030A-archless} mutant and slow growth is observed for the Mtr4^{E1033A-archless} mutant.