Figure 6.
Specific alleles of RSP5 differentially affect pCPS entry into the MVB pathway and ubiquitination of Ste2. (A) Localization of GFP-CPS in mvb326 cells, harboring a plasmid encoding HA-Rsp5 (top), or rsp5Δ cells harboring a plasmid encoding Rsp5-326 (bottom), at 26°C. (B) SEY6211 (wild-type) and SSY22 (mvb326) cells expressing Ste2-HA were treated with alpha factor for 8 min at the indicated temperatures and lysates were prepared and probed with anti-HA antibody. Unmodified Ste2, phosphorylated Ste2, and ubiquitinated Ste2 are indicated by brackets. (C) CPS was immunoprecipitated from either CBY16 (pep12Δ) or DKY83 (smm1 pep12Δ) cells, grown at 26°C, followed by Western blot analysis by using anti-ubiquitin antibody. (D) LH291 (wild-type), LH23 (rsp5-1) MYY290 (wild-type) and MYY833 (mdp1-13) cells were incubated at 37°C for 10 min, before labeling with Trans 35S-label for 10 min at 37°C. Cells were treated as described in Figure 2. An asterisk marks a nonspecific band as described in Figure 2.