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.
Copyright (c) 2010 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cluster Analysis
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F-Box Proteins / genetics
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F-Box Proteins / metabolism
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GTP-Binding Protein alpha Subunits, Gq-G11 / genetics
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GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
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GTP-Binding Protein beta Subunits / genetics
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GTP-Binding Protein beta Subunits / metabolism
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GTP-Binding Proteins / genetics
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GTP-Binding Proteins / metabolism*
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Gene Expression Profiling
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Gene Expression Regulation, Fungal
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Genome, Fungal
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Humans
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Phenotype
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Pheromones / genetics
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Pheromones / metabolism
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Promoter Regions, Genetic
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Reproducibility of Results
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SKP Cullin F-Box Protein Ligases / genetics
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SKP Cullin F-Box Protein Ligases / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Signal Transduction / genetics*
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Substrate Specificity
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes / genetics
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Ubiquitin-Protein Ligase Complexes / metabolism
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
Substances
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CDC4 protein, S cerevisiae
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Cell Cycle Proteins
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F-Box Proteins
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GTP-Binding Protein beta Subunits
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Pheromones
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Ste4 protein, S cerevisiae
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CDC34 protein, S cerevisiae
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CDC34 protein, human
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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SKP Cullin F-Box Protein Ligases
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Ubiquitin-Protein Ligases
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GTP-Binding Proteins
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GPA1 protein, S cerevisiae
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GTP-Binding Protein alpha Subunits, Gq-G11