Figure 1.

Rsp5p is required for Ste2p ubiquitination and internalization. (A) α-Factor internalization assays performed by the continuous presence protocol at 37°C (see MATERIALS AND METHODS). Cells were propagated in SD medium at 24°C and shifted to 37°C for 15 min before the addition of radiolabeled α-factor. RSP5 (LHY434, ♦), rsp5-2 (LHY433, ⋄), RSP5 (LHY291, ●), rsp5–1 (LHY23, ○). (B) Ste2p modification before and after α-factor (αF) treatment. ubc1Δ ubc4Δ (LHY1394), end4-1 (LHY501), rsp5-2 (LHY433), wild-type (LHY434), and ste2Δ (LHY10) cells were treated (+) or not (−) with 1 μM α-factor for 8 min at 37°C. Total cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Ste2p antibodies. Phosphorylated (P) and monoubiquitinated (Ub) receptor species are indicated by the labeled brackets.