FIG. 1.

Rcy1p is localized in areas of polarized growth in an actin-dependent manner. (A) JMG201 cells (rcy1::GAL-GFP-RCY1) were grown to early log phase at 30^°C in selective medium. Cells were analyzed by fluorescence (upper panels) and phase-contrast (lower panels) microscopy as described in Materials and Methods. (B) Upper panel, JMG201 cells were grown as described for panel A (overexpression [OE] of GFP-Rcy1p). Four hours after addition of galactose, α-factor (50-μg/ml final concentration) was added and left for 2 h, and GFP-Rcy1p was visualized by fluorescence microscopy. Lower panel, wild-type cells (K699) were transformed with a plasmid expressing GFP-Rcy1p from its own promoter (end) and treated with α-factor for 2 h. (C) JMG201 cells were grown as described above and either treated (lower panel) or not treated (upper panel) with α-factor. After 2 h, LAT-A was added and left for 5 min, the cells were fixed, and the localization of GFP-Rcy1p was visualized. (D) The expression of GFP-Rcy1p in JMG201 cells shown in panels A (lane 2, as), B (lane 3, α-factor), and C (lane 4, LAT-A) was analyzed by immunoblotting with polyclonal antibodies against GFP. Cells with an empty control vector were used as a control (lane 1, −). Note that neither pheromone treatment nor addition of LAT-A alters GFP-Rcy1p levels. (E) The localization of GFP-Rcy1p expressed from the GAL promoter was examined in sec18-1 (RH144) or end4Δ (RH1965) cells by GFP microscopy. sec18-1 cells were incubated at the restrictive temperature (37°C) for 1 h before analysis of the localization of Rcy1p-GFP.