Figure 2.
Effect of ubiquitin overexpression on Ste6 turnover in the doa4 mutant. Ste6 half-life was determined by pulse-chase experiments in the doa4 strain JD116 and the wild-type strain JD52 transformed with the ubiquitin-overexpressing plasmid YEp96 or the control vector YEplac112. Cells grown at 30°C in the presence of 100 μM CuSO4 were labeled for 15 min with Tran35S-label (ICN) and were then chased with an excess of cold methionine and cysteine. Ste6 was immunoprecipitated from cell extracts prepared at time intervals as indicated. The precipitated proteins were analyzed by SDS-PAGE and autoradiography. (A) From top to bottom: JD116 (doa4)/YEplac112 (vector), JD116 (doa4)/YEp96 (CUP1-UBI), JD52 (wt)/YEplac112 (vector), JD52 (wt)/YEp96 (CUP1-UBI). The Ste6 band is marked with an arrow; a background band is marked with an asterix. (B) Signal intensities (logarithmic scale) plotted against the chase time. ▪, JD116/YEplac112; □, JD116/YEp96; ●, JD52/YEplac112; ○, JD52/YEp96.