Abstract
In the Saccharomyces cerevisiae pheromone response pathway, the Gbetagamma complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that Gbetagamma must remain membrane-associated after release from Galpha to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires Gbetagamma function and the Gbetagamma-binding domain of Ste5, but not the kinases downstream of Gbetagamma, suggesting that it is mediated by Gbetagamma itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gbetagamma. Remarkably, although independent of Gbetagamma, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gbetagamma. Thus, membrane recruitment of Ste5 defines a molecular activity for Gbetagamma. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Amino Acid Sequence
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Carrier Proteins*
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Cell Cycle Proteins / metabolism
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Crosses, Genetic
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Fungal Proteins / metabolism*
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GTP Phosphohydrolases / metabolism
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GTP-Binding Proteins / chemistry
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GTP-Binding Proteins / metabolism*
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Genotype
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Guanine Nucleotide Exchange Factors*
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Intracellular Signaling Peptides and Proteins
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MAP Kinase Kinase Kinases
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Mating Factor
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Molecular Sequence Data
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Peptides / genetics
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Peptides / physiology*
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Pheromones / physiology*
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins / metabolism
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Recombinant Fusion Proteins / biosynthesis
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / physiology*
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Saccharomyces cerevisiae Proteins*
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Sequence Alignment
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Sequence Homology, Amino Acid
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beta-Galactosidase / biosynthesis
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beta-Galactosidase / genetics
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
Substances
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Adaptor Proteins, Signal Transducing
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CDC24 protein, S cerevisiae
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Carrier Proteins
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Cell Cycle Proteins
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Fungal Proteins
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Guanine Nucleotide Exchange Factors
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Intracellular Signaling Peptides and Proteins
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Peptides
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Pheromones
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Proto-Oncogene Proteins
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Recombinant Fusion Proteins
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STE5 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Mating Factor
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Protein Serine-Threonine Kinases
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MAP Kinase Kinase Kinases
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STE20 protein, S cerevisiae
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beta-Galactosidase
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GTP Phosphohydrolases
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GTP-Binding Proteins
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae