Systematic analysis of essential genes reveals important regulators of G protein signaling

Abstract

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways.

Figure 1. Validation of the TetO₇ Promoter Essential Gene Screen

(A) Transcriptional activation (β-galactosidase activity) in response to α factor treatment was measured spectrofluorometrically in TetO₇-GPA1 cells treated with doxycycline (Dox, 10ng/mL and 100ng/mL) or untreated control. Cells were transformed with a plasmid containing the pheromone-inducible reporter FUS1-lacZ. Data were analyzed by non-linear regression (sigmoidal-dose response, variable slope) using GraphPad Prism software. Results are the mean ± S.E. for three individual experiments each performed in triplicate. (B) TetO₇-CDC42 cells treated as in (A). (C) TetO₇-WT cells treated as in (A). (D) Percentage of essential genes associated with the indicated GO Process. (E) Percentage of essential gene hits associated with the indicated GO Process. (F) Fold-enrichment of hits compared to all essential genes for each GO Process.

Figure 2. Phenotype Clustering Analysis

Gene hits were analyzed by Cluster 3.0 software based on maximum response, EC50, and basal activity normalized to the untreated control and converted to Log₂. Gene similarity was calculated using Pearson correlation (uncentered correlation) and clusters were generated using centroid linkage. Clustering data was visualized by Java TreeView (v 1.1.3). Genes were labeled by their involvement in the indicated GO Process. See also Figure S1 and Table S1.

Figure 3. Verification of the Roles of Selected Essential Genes in Pheromone Signaling

(A–G) The indicated essential genes were chosen for further validation and analysis. TetO₇ strains expressing FUS1-lacZ were treated with 10µg/mL doxycycline for 15hrs and exposed to the indicated concentrations of α factor for 90min. Below each pheromone dose-response curve is a corresponding immunoblot probed using phospho-p42/44 (P-Fus3, P-Kss1) or G6PDH (load control) antibodies. TetO₇ strains were treated with 10µg/mL doxycycline for 15hrs and then 3µM α factor for 30min. Results are the mean ± S.E. (n=5). See also Figure S3.

Figure 4. The Cdc34 E2 and SCF^Cdc4 Regulate Signaling Upstream of Ste4^Gβ

(A) TetO₇-CDC4 cells were transformed with a plasmid containing either STE11-4 (constitutively active mutant) or no insert. Cells were treated with 10µg/mL doxycycline for 15hrs and then 3µM α factor for 30min. Samples were analyzed by immunoblotting using phospho-p42/44 or G6PDH antibodies. Bar graphs represent quantification of the indicated bands. Results are the mean ± S.E. (n=3). (B) TetO₇-CDC4 cells were transformed with a plasmid containing STE4 under the control of a galactose-inducible promoter. Cells were treated with 10µg/mL doxycycline for 12hrs in medium containing either dextrose or switched to galactose (2% w/v final concentration) for 3hrs prior to α factor treatment (3µM for 30min). Samples were analyzed by immunoblotting using phospho-p42/44 or G6PDH antibodies. Bar graphs represent quantification of the indicated bands. Results are the mean ± S.E. (n=3).

Figure 5. SCF^Cdc4 Ubiquitinates Gpa1 in vitro and Facilitates its Turnover in vivo

(A) Gpa1 stability in wild-type vs. temperature-sensitive cdc4-1 mutant cells. Cultures were grown at 25°C, shifted to 37°C for 1hr, and then treated with cycloheximide (CHX) for the indicated times. Myristoylated (bottom band) and unmyristoylated (top band) Gpa1 detected by immunoblotting with Gpa1 antibodies. (B) Samples from panel (A) analyzed with Sst2 antibodies. (C and D) The intensity of bands from (A) and (B), analyzed by densitometry. Results are the mean ± S.E. (n=3). (E) In vitro ubiquitination of Gpa1. Purified Gpa1-Flag was incubated with purified SCF^Cdc4 complex (Flag-Skp1/Cdc53/Myc-Rbx1/Cdc4), His6-Uba1, His6-Cdc34, and ubiquitin as indicated, followed by SDS-PAGE and immunoblotting. Unmodified (Gpa1) and ubiquitinated (Gpa1-(Ub)_n) Gpa1 protein was visualized with Gpa1 antibodies. Membranes were also probed with Cdc4 antibodies and Cdc53 antibodies. (F) In vitro ubiquitination using Lys-less ubiquitin (Ub^0K). Reactions contain either Gpa1 or Gpa1^Δ128-236, a mutant form of Gpa1 lacking the ubiquitinated subdomain. (G) In vitro ubiquitination of Gpa1 using Ub^0K and SCF complexes containing either Cdc4 or Met30 as indicated. Note that Met30 appears to bind weakly to Gpa1 but does not sustain Gpa1 ubiquitination (Figure S4B). See also Figure S4.

Figure 6. The Pheromone Response Pathway

The Cdc34/SCF complex targets several known substrates in the pheromone response pathway. Known substrates of Cdc34/SCF are shown in dark grey. Likely substrates of the SCF are shown in light grey. SCF^Cdc4 substrates that have been verified using in vitro ubiquitination assays are designated with an asterisk (*).