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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., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.