Fig. 2.

Sx16 complements mutant phenotypes of tlg2Δ yeast. The fidelity of the endocytic system of tlg2Δ yeast (NOzY3/4) producing HA-tagged Tlg2p, Sx16 or Sx4 as in Fig. 1 or carrying the empty vector pNB701 was assessed. (A) Aliquots of 5 μl of growing cultures (and tenfold serial dilutions thereof) were spotted onto SD –ura +50 μM CuCl₂ plates, containing 1.5 M KCl (high salt) or not (control), and incubated at 30°C for 2-3 days. (B) Delivery of Ste3p-Myc to the vacuole was monitored by following its degradation in the cells described above that also produced Ste3p-Myc from the GAL1 promoter. Ste3p expression was induced during a 3-hour period in galactose medium. The `chase' was initiated by the addition of glucose to 3% (w/v). The initial (0) time point was taken here with subsequent time points taken as indicated (minutes after glucose addition). The amount of Myc-tagged Ste3p at each time point was analysed through immunoblotting (the same filters were also probed for Vph1p to control for equal loading). (C) Uptake of FM4-64 from the cell surface was followed by labelling cells at 0°C and initiating a chase period through incubation in fresh, pre-warmed media for 10 or 45 minutes as indicated. Representative images of the staining displayed by the majority of cells at each time point are shown. Quantification of data obtained from three experiments of this type is shown in Table 1. Scale bars: 2 μm.