FIGURE 2.
Phosphorylation may be required for Ste4 ubiquitination. Cells expressing either wild-type Ste4 or Ste4T322A/S335A were transformed with either an empty vector or a plasmid 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.