Figure 3.

Ligand-mediated delivery of Ste2 to the vacuole is abolished in snc cells and SNC1^ala43 cells shifted to restrictive temperatures. (A) Delivery of Ste2-GFP, but not Ste2Δ-GFP, to the vacuole occurs in snc cells grown on galactose. snc cells (JG8) expressing either Ste2-GFP (snc STE2) or a C-terminal truncated form of Ste2-GFP (snc STE2Δ), which does not undergo endocytosis, were grown to log phase at 26°C before labeling with FM4–64 (see MATERIALS AND METHODS). Cultures were split, whereby half were mounted in soft agar containing no additions, while the other half were mounted in agar containing α-factor (1 μM) and were processed immediately for microscopy. A composite image of untreated cells is representative of the zero time (0 min), while specific α-factor–treated cells were monitored as a function of time. (B) Delivery of Ste2-GFP to the vacuole is abolished in snc cells grown on glucose. snc cells (JG8) expressing SNC1 constitutively or bearing a control plasmid were transformed with plasmids expressing Ste2-GFP to give SNC1 STE2 and snc STE2 strains, respectively. In addition, snc cells expressing the truncated form of Ste2-GFP (snc STE2Δ) also were used. Cells were grown to log phase at 26°C, before labeling with FM4–64. Next, samples of cells were mounted in agar containing no additions, while an equal amount was mounted in agar containing α-factor (1 μM) before visualization, as described above. (C) Delivery of Ste2-GFP to the vacuole is abolished in SNC1^ala43 cells shifted to the restrictive temperature. snc cells (JG8) expressing SNC1^ala43 were grown to log phase and either incubated with FM4–64 at 26°C or shifted for 30 min to 37°C before labeling with FM4–64 at the restrictive temperature. Cells that were treated with α-factor (1 μM) were monitored over time by microscopy.