Figure 3.

Mutation of Chs3 residues 19–21 causes a defect in internalization. (A) Chs3-GFP plasmids containing alanine scanning mutants were transformed into a chs3ΔSEC7-Mars strain and plated on indicated concentrations of CW. Plates were imaged after 2 days at 30°C. See also Figure S2 for additional CW concentrations. (B) Protein expression level of alanine-scanning Chs3-GFP constructs relative to wild type as calculated from the α-GFP Western blots in Figure S1, normalized to loading control. Mutations that confer CW resistance phenotypes are marked with (+). (C) GFP and Sec7-Mars localization in chs3Δ cells for WT Chs3-GFP and Chs3(₁₉DEE₂₁-→ AAA)-GFP, at equivalent light levels. Scale bar, 1 μm. (D) Line profile of intensity along dotted line in (C) of unbudded cell expressing mutant protein, or across non-bud section of cell expressing WT protein. Asterisks mark higher localization of Chs3(₁₉DEE₂₁ → AAA)-GFP to the plasma membrane at cell boundaries (dotted lines). (E) Chs3(Δ10–27)-GFP and Chs3(₁₉DEE₂₁ → AAA)-GFP were introduced into the chs3Δ and chs3Δapl2Δ strains and plated on indicated media. Plates were imaged after 2 days at 30°C. See also Figure S2 for additional CW concentrations.