Ppz phosphatases regulate the phosphorylation and activity of Art1. (A) Mup1-pHluorin–trafficking assays were performed to characterize endocytic trafficking in the indicated yeast strains. ART1 is expressed from its native promoter on a pRS416 centromere plasmid and contains a C-terminal 3× FLAG fusion. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (B) SILAC-based quantification of phosphorylation events was performed (according to the scheme shown in Fig. S1 A) using endogenous Art1-FLAG as bait. Color coding of regions of Art1 corresponds to the schematic representation of Art1 (Fig. 1 E). Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from three experiments (n = 3), except for S85, S106, S722, and S798, which were only resolved and quantified in two replicate experiments. (C) The indicated yeast strains (SEY6210 background) expressing empty vector or the indicated Art1 expression plasmid were plated on minimal media (SCD-ura) containing the indicated concentration of canavanine. (D) Mup1-pHluorin–trafficking assays were performed to characterize endocytic trafficking in the indicated yeast strains with Art1 variants expressed from a pRS416 centromere plasmid (as in Fig. 4 A and Fig. S1 A). Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (E) The indicated yeast strains (SEY6210 background) expressing empty vector or the indicated Art1 expression plasmid were plated on minimal media (SCD-ura) containing the indicated concentration of canavanine.