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. 2011 Jan 24;192(2):229–242. doi: 10.1083/jcb.201008121

Figure 2.

Figure 2.

Cells lacking Rsp5 activity still sort ubiquitinated cargo proteins into vacuoles. (A) Wild-type and temperature-sensitive rsp5-1 cells expressing HA-Vps27 and Myc-Ub were shifted to 37°C for 1 h. Input lysates and Vps27 immunoprecipitates (IP) were immunoblotted (IB) for HA (Vps27) and Myc (Ub). Twice the amount of the anti-HA IP from rsp5-1 cells was loaded. *, expected molecular weight; **, ubiquitinated forms. (B) Localization of Mup1-GFP and Mup1-GFP-Ub in mutant EN67 cells lacking multiple Rsp5 adaptors. Cells were grown in YPD and copper for 4 h to produce Mup1 fusion proteins from the CUP1 promoter. (C) Sorting of Gap1-GFP, Mup1-GFP, and Mup1-GFP-Ub after Rsp5 inactivation. Cells (rsp5-1) were shifted to 37°C for 1 h and grown in YPD in the presence of 100 µM CuCl2 to induce production of GFP-tagged proteins under control of the CUP1 promoter. (D) Localization of Gap1-GFP, Ste3-GFP, Mup1-GFP, Ste3-GFP-Ub, or Mup1-GFP-Ub in rsp5Δ-null cells grown in YPD + 25 µM CuCl2. (E) Localization of Mup1-GFP or Mup1-GFP-UbK63R in cells expressing wild-type (WT) Ub (SUB492) or K63R Ub (SUB493) as their sole source of Ub. Bars, 5 µm.