FIGURE 5.
Lys-340 is required for pheromone-induced ubiquitination of Ste4. Cells expressing either wild-type Ste4 or Ste4K340R were transformed with either an empty vector or plasmids expressing His-8-tagged ubiquitin (His8-Ubi). Early log phase cells were treated with 3 μm pheromone for 60 min. His-8-Ubi-conjugated proteins were purified from whole cell lysates under denaturing conditions. The level of Ste4 in the whole cell extracts (WCE) prior to purification was detected by immunoblotting (IB) with anti-Ste4 antibodies (top panel). The purified samples were analyzed by immunoblotting with anti-Ste4 antibodies (center panel) and anti-ubiquitin antibodies (bottom panel). p-Ste4, phosphorylated Ste4; Ubi-Ste4, ubiquitinated Ste4.