FIGURE 7:

Absence of α-arrestins Ldb19, Rod1, and Rog3 delays internalization and delivery of endocytosed FAP-Ste2 to the vacuole. (A) Otherwise isogenic MATa (yAEA380) and MATa 3arr∆ (yAEA381) cells expressing FAP-Ste2 and Vph1-EGFP were cultivated and incubated with 5 μM α-factor to initiate pheromone-induced endocytosis as described in Figure 6A. A representative image is shown for each strain at each time point. Scale bar, 5 μm. (B) The data in A were quantified and plotted as described in Figure 6B. The initial intensities of FAP-Ste2 on the PM (i.e., at time 0) were quite similar for both strains, and their median values were set to 100%. (C) MATa yps1Δ mkc7Δ cells expressing either FAP-Ste2 (yAEA359) or FAP-Ste2(7K-to-R) (yAEA397) were grown at 20 °C to early exponential phase, treated with LatA, incubated with fluorogen (0.4 mM dye; 15 min; pH 6.5), and deposited onto the glass bottoms of imaging chambers, and then internalization was initiated by washing out the LatA and excess fluorogen, as described under Materials and Methods, followed by immediate addition of α-factor in H₂O (5 μM final concentration), and the cells were monitored by fluorescence microscopy at the indicated times over the course of 60 min. A representative image is shown for each time point. Scale bar, 5 μm.