FIG 7.

Ubiquitination is essential for formation of Rim20 foci. (A) YOK3342 (RIM20-EGFP) and YOK3343 (rsp5-1 RIM20-EGFP) were grown to log phase at 30°C, further cultured for 90 min at 30°C or 38°C, and exposed to alkaline medium at 30°C or 38°C. After 60 min of alkaline treatment, cells were subjected to fluorescence microscopy. (B) YOK3403 (AID-RSP5 RIM20-EGFP) cells in log phase were pretreated with 500 μM IAA or ethanol (mock) for 2 h, exposed to alkaline medium for 60 min, and subjected to fluorescence microscopy. (C) YOK3412 (RIM8-2×EGFP) and YOK3413 (AID-RSP5 RIM8-2×EGFP) cells in log phase were pretreated with 500 μM IAA or ethanol (mock) for 2 h, exposed to alkaline medium for 20 min, and subjected to fluorescence microscopy. (D) YOK3413 cells were cultured as described for panel C, and total cell lysates were prepared at each time point. Immunoblotting was performed with an anti-AID or, to demonstrate uniform protein loading, anti-Pgk1 antibody.