Ste4 undergoes ubiquitination in response to pheromone stimulation. Wild-type or ste4Δ cells were transformed with either empty vector or a plasmid expressing His-8-tagged ubiquitin (His8-Ubi). Early log phase cells were treated with 3 μm pheromone for the indicated time (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.

Phosphorylation may be required for Ste4 ubiquitination. Cells expressing either wild-type Ste4 or Ste4^T322A/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.

Rsp5 is required for Ste4 ubiquitination in vivo. A, Ste4 coimmunoprecipitates with Rsp5. Wild-type or ste4Δ cells were transformed with either an empty vector or a plasmid expressing FLAG-tagged Rsp5 (Rsp5-FLAG). Early log phase cells were treated with 3 μm pheromone for 1 h. FLAG-tagged Rsp5 was immunoprecipitated (IP) using anti-FLAG M2 resin, and the copurified Ste4 was detected by immunoblotting of the immunoprecipitates (IP: Flag) using anti-Ste4 antibodies. The relative level of input Ste4 and Rsp5-FLAG in the whole cell extracts (WCE) prior to purification is shown in the lower panels. B, turning off RSP5 expression abolishes Ste4 ubiquitination. Wild-type (TetO₇-WT) or TetO₇-RSP5 (RSP5 under the control of doxcyclin-regulatable promoter) cells were transformed with either an empty vector or a plasmid expressing His-8-tagged ubiquitin (His8-Ubi). Early log phase cells were treated or not treated with 60 μg/ml doxycycline (Dox) for the indicated time (h), followed by treatment of 3 μm pheromone for 1 h. 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.

Rsp5 ubiquitinates Ste4 in vitro. Bead-immobilized Ste4-FLAG was incubated with Rsp5 or catalytically inactive Rsp5 (Rsp5-C/A) in the absence or presence of E1 activating enzyme (Uba1). All reactions contained Ubc5b, ATP, and ubiquitin. Reactions were stopped after 30 min with 2% SDS. Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with M2 anti-FLAG antibody and goat-anti-mouse-HRP secondary antibody. Monoubiquitination slows the electrophoretic mobility of Ste4-FLAG by the approximate mass of ubiquitin (∼8 kDa). Long (top panel) and short exposures (center panel) are shown. After immunoblotting, the nitrocellulose blot was stained with Coomassie to show Rsp5 autoubiquitination and Uba1 loading (bottom panel). *, antibody heavy chain. Because we observed that FLAG peptide elution buffer inhibited Rsp5 catalytic activity, we conducted in vitro ubiquitination with bead-immobilized Ste4-FLAG, which may also reduce reaction efficiency.

Lys-340 is required for pheromone-induced ubiquitination of Ste4. Cells expressing either wild-type Ste4 or Ste4^K340R 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.

Blocking ubiquitination does not alter the stability of Ste4. A, cells expressing either wild-type Ste4 or Ste4^K340R were grown to early log phase, treated with 3 μm pheromone for 1 h, and followed by treatment with the protein synthesis inhibitor cycloheximide for the indicated times. The level of Ste4 and Ste4^K340R was detected by immunoblotting (IB). B, early log TetO₇-RSP5 (RSP5 under the control of doxycycline-regulatable promoter) cells were first not-treated (-Dox) or treated with doxycycline (+Dox) for 9 h (to switch off RSP5 expression in the TetO₇-RSP5 cells). Cells were then treated with 3 μm pheromone for 1 h, followed by treatment with the protein synthesis inhibitor cycloheximide for the indicated times. The abundance of Ste4 was detected by immunoblotting (IB). Equal loading of each lane was confirmed by immunoblotting with anti-Pgk1. Note that Ste4 is dephosphorylated more prominently in the TetO₇-RSP5 strain, which resulted in the appearance of two bands.

Ubiquitination of Ste4 does not regulate the magnitude and duration of pheromone-induced activation of MAP kinases. A, whole cell extracts were prepared from the wild type and the Ste4^K340R mutant treated with 3 μm pheromone α factor for the indicated time, resolved by 10% SDS-PAGE, and probed with anti-phospho-p44/42 (top panel) or anti-Ste4 (bottom panel) antibodies. p-Mpk1, p-Kss1, and p-Fus3 denote phosphorylated and thus activated Mpk1, Kss1, and Fus3. B, pheromone-dependent transcriptional induction was measured following transformation of the above cells with a pheromone-responsive FUS1 promoter-GFP reporter. Cells were then treated with 3 μm α-factor for 90 min, and the resulting fluorescence in each cell was monitored by cell sorting. Pathway activation results in an increase in cells with >40 fluorescence units of activity (as indicated by an M1 bar on each graph).

Ubiquitination of Ste4 limits pheromone-induced polarized growth. Cells expressing either wild-type Ste4 or Ste4^K340R were grown to A₆₀₀ 0.2 and treated with dimethyl sulfoxide (1%, final concentration) for 1.5 h before the addition of nocodazole (15 μg/ml in 1% dimethyl sulfoxide, final concentrations) for 2.5–3 h. Cultures were visualized by microscopy to confirm G₂/M arrest. The cells were then loaded into a microfluidics device, and the nocodazole was washed out for 20 min prior to stimulation with 150 nm α factor. Cells were imaged at 5 min intervals for 2 h. For each cell, the time at which the first sign of polarized growth detectable in the differential interference contrast image was recorded and plotted as a function of time. The slope was taken from the linear portion of each curve, and the average slopes from three independent experiments are shown in the bar graph (right panel). The difference between Ste4 and Ste4^K340R was statistically analyzed by t test. *, p < 0.05). Error bars, mean ± S.E.