Mapping of a yeast G protein betagamma signaling interaction.
The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.
Dowell
S J
SJ
Glaxo Wellcome Research and Development, Stevenage, SG1 2NY, United Kingdom. sd14041@glaxowellcome.co.uk
Bishop
A L
AL
Dyos
S L
SL
Brown
A J
AJ
Whiteway
M S
MS
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002352:Carrier Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C084294:STE18 protein, S cerevisiae; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D019204:GTP-Binding Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins