FIGURE 3:

The predicted YxxΦ-binding pocket is required for some but not all functions of Apm1 and Apm2. (A) Expression of an Apm1 mutant lacking a functional tyrosine signal-binding pocket (apm1^tyr) or overexpression of APM2 from the APM1 promoter (APM2^OE) is unable to restore the vacuolar localization of the AP-1–specific cargo Sna2^Y75A-GFP in apm1 mutant cells. Scale bar, 2 μm. (B) Quantification of surface fluorescence of Sna2^Y75A-GFP. Error bars represent SEM (n = 3). Unpaired t test compared with apm1Δ, ***p < 0.0001 and **p < 0.01. (C) An Apm2 mutant lacking a functional tyrosine signal-binding pocket (apm2^tyr) and expressed on a plasmid from its native promoter exhibits sertraline sensitivity. Yeast were plated in a 10× dilution series on YPD containing Hyg B and DMSO or 22.5 μM sertraline. (D) The apm2^tyr mutant is unable to restore sorting of the Snc1 reporter GSS. Cell-surface GSS levels in the indicated strains were determined by quantifying invertase activity and normalizing to levels in wild-type cells. Unpaired t test compared with wild type, ***p < 0.0001. Error bars represent SEM (n = 6). (E) Wild-type APM1 and apm1^tyr, but not overexpressed APM2 (APM2^OE), restores the calcofluor resistance of chs6 apm1 mutants. Yeast were plated in a 10× dilution series on buffered YPD containing Hyg B and 50 μg/ml calcofluor white (CW). (F) Representative fluorescence images of calcofluor white–stained strains.