Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway.

Abstract

The Saccharomyces cerevislae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein a, ,, and y subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPAI, STE4, and STE18, using the galactose-inducible GAL] promoter. Overexpression of STE4 alone, or STE4 together with STEJ8, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUSI 25-to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa!a diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a stel8 null strain. GPAI hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPAI were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPAI may function in an adaptation response to pheromone.G proteins function as molecular transducers of extracellular stimuli, coupling these stimuli to intracellular responses (reviewed in references 12 and 48). G-protein-mediated signal transduction has now been described in a number of eucaryotes as phylogenetically diverse as fungi and vertebrates. Elements from several of these systems have been biochemically characterized in some detail, and they appear to conserve many aspects of structure and function. In the best-understood pathways, in mammalian cells, G proteins transduce a signal generated by stimulation of a membrane receptor of the rhodopsin/lB-adrenergic family (10, 36). These G proteins, consisting of a, 1, and y subunits, exist as inactive heterotrimers with GDP bound to the a subunits. Receptor stimulation leads to exchange of GDP for GTP, activating the G protein, which then dissociates into a and P-y subunits. Both a and fly subunits are then capable of interacting with diverse intracellular effectors in different systems (reviewed in reference 37), including adenylate cyclase, cyclic GMP phosphodiesterase, and phospholipase A2. Hydrolysis of GTP to GDP inactivates the a subunit, which then reassociates with free P-y to return to the inactive aIly configuration.

The Cdc6 Protein Is Ubiquitinated in Vivo for Proteolysis in Saccharomyces cerevisiae

“…15Dau is a MATa derivative of BF264-15D (18). Temperature-sensitive and deletion mutants used were as follows: cdc4-1 leu2 ura3; cdc4-1 GAL1: CDC6(URA3) leu2 ura3; cdc4-1 sic1::URA3 leu2 ura3; cdc34-2 ura3 his3; cdc34 -2 sic1::URA3 leu2 ura3; and cdc6::kanMX4 GAL1:CDC6 (URA3).…”

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…Integration of the mutant allele into the chromosome eliminates variability in plasmid copy number that affects protein expression levels. The pheromone response pathway is exquisitely sensitive to changes in Gpa1p expression (Cole et al, 1990;Kang et al, 1990). Studies of other GPA1 mutations have revealed different phenotypes when expressed on a centromere-based plasmid instead of the chromosomal locus (Miyajima et al, 1989;Kurjan et al, 1991).…”

PsTRXh1 and PsTRXh2 Are Both Pea h-Type Thioredoxins with Antagonistic Behavior in Redox Imbalances

“…Plant TRX expression in yeast cells was achieved using either the inducible Ycp2 shuttle vector (Cole et al, 1990) or the constitutive pFL61 vector (Minet et al, 1992). Each plant TRX was amplified from the corresponding full-length cDNA by PCR using specific pairs of oligonucleotides designed to introduce either MluI (5#)/BamHI (3#) or NotI (5#)/NotI (3#) sites necessary for cloning into Ycp2 and pFL61 vectors, respectively (MluIhN/PeahC and NotIhN/NotIhC to amplify and clone h1 ORF; MluIh2N/Peah2C and NotIh2N/NotIh2C for h2 cloning; Table I).…”