Mapping of a Yeast G Protein βγ Signaling Interaction

Abstract

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 βγ subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gβ (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 Gβγ coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Gα) and Ste18p (Gγ) 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 Gβγ coiled-coil in Ste5p binding may set a precedent for Gβγ-effector interactions in more complex organisms.

Dynamic Studies of Scaffold-Dependent Mating Pathway in Yeast

“…Another effector of Gβγ is the scaffold protein Ste5. The correlation between the disruption of the Ste4(Gβ)-Ste5 interaction and sterility confirms the importance of this interaction in signal transduction (29). Gβγ can bind to Ste5 on the LIM domain of Ste5, which is required for Ste11 (MAPKKK) activation (30), probably through inducing a conformational change that enhances Ste20-dependent activation of Ste11.…”

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus

“…After 1 h at 30°C, chemiluminescence was determined in a Top‐count scintillation counter (Packard). Assays using the substrate ONPG were performed as described by Dowell et al . (1998).…”

“…The lys2Δ::FUS1-lacZ disruption cassette was produced by PCR from YIpFUS102 (Nomoto et al, 1990) using primers TATTTAAATT-ATTGTACATGGACATATCATACGTAATGCTCAACCGAAATGG-ATACGGATAAGTTAATC and AAACTGCTAATTATAGAGAG-ATATCACAGAGTTACTCACTATTATTATTATTTTTGACACCAG-ACC. NSY1 was generated by transforming lys2Δ::FUS1-lacZ into 2762 MMY8, selecting on α-aminoadipic acid and screening by a blue colony assay as described by Dowell et al (1998) for integrants in which the pheromone response pathway was activated.…”

Combinatorial phosphorylation modulates the structure and function of the G protein γ subunit in yeast

“…However, evidence suggests that the N-terminal regions of G and G, which are proximal to each other in the G dimer, play an important role in Gdependent effector signaling. In the budding yeast Saccharomyces cerevisiae (yeast), the effector-binding sites on G are located in N-terminal residues in G Ste4 (14,15), which overlap with the same region in human G 1  2 that inhibits phospholipase C 2 activity in mammals (16). Mutation near this region modulates mammalian G-dependent activity of adenylyl cyclase 5 (AC5) and AC6 in vitro (17).…”