FIG 5.

Rsp5-mediated ubiquitination is essential for the Rim101 pathway. (A) YPH499 (WT) and FAY51R (rsp5-1) cells harboring pFI1 (HA-RIM101) were grown to log phase at 30°C and then further cultured for 90 min at 30°C or 38°C. Cells were then exposed to alkaline medium at 30°C or 38°C for 20 min. Total cell lysates were subjected to immunoblotting with an anti-HA or, to demonstrate uniform protein loading, anti-Pgk1 antibody. FL and ΔC, full-length and processed Rim101, respectively. (B) YOK3073 (WT), YOK3094 (rim21Δ), and YOK3393 (AID-RSP5) cells harboring pFI1 grown to log phase were pretreated with 500 μM IAA or ethanol (mock) for 2 h and then exposed to alkaline medium for 20 min. Total cell lysates were subjected to immunoblotting with an anti-HA, anti-AID, or, to demonstrate uniform protein loading, anti-Pgk1 antibody. FL and ΔC, full-length and processed Rim101, respectively. (C) YOK2649 (RIM8-HA) and YOK2658 (RIM8-HA doa4Δ) cells in log phase were exposed to alkaline medium for 20 min. Total cell lysates were subjected to immunoblotting with an anti-HA or, to demonstrate uniform protein loading, anti-Pgk1 antibody. Ub, ubiquitin.