Activated Alleles of the Schizosaccharomyces pombe gpa2 ⁺ Gα Gene Identify Residues Involved in GDP-GTP Exchange

Abstract

The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 G␣ subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 G␤␥ dimer to repress transcription of the glucose-regulated fbp1 ؉ gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2 ؉ chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in G␣ function, thus extending our understanding of G␣ protein structure-function relationships.Heterotrimeric guanine-nucleotide binding proteins (G proteins) relay extracellular signals from surface receptors to intracellular effectors. G proteins are composed of G␣ subunits and G␤␥ dimers whose interaction depends upon whether G␣ is bound to GDP or GTP. The inactive heterotrimer contains a G␣ subunit bound to GDP. Ligand binding by seven transmembrane G protein-coupled receptors (GPCRs) leads to GDP release and GTP binding by G␣, resulting in a conformational change that decreases G␣ affinity for G␤␥ and, depending on the system, promotes effector binding (32). Either or both G␣-GTP and G␤␥ are then able to regulate downstream effectors (28). Restoration of the basal state is achieved through hydrolysis of the bound GTP by the G␣ and G protein heterotrimer reassociation (28). GTP hydrolysis can be accelerated by protein-protein interactions of G␣ with regulator of G protein signaling (40, 50) proteins or through G␣-effector interactions (4).A variety of approaches, including mutational studies, biochemical analyses of purified G protein subunits, and highresolution crystallography, inform our current understanding of the mechanisms behind guanine nucleotide binding and release, as well as GTP hydrolysis reactions that govern G␣ activation and inactivation cycles. Crystal structure data are available for G␣ subunits in the GDP-bound (20, 31) or GTPbound (5, 29, 42) conformation. G␣ subunits possess two domains, a helical domain and a GTPase domain that resembles small GTPases, such as Ras, Rho, and Rheb. Bound guanine nucleotides are buried in a cleft between these domains, which are connected by two linker sequences. Current models suggest that interdomain interactions regulate both receptor-activated and basal GDP release rates (8,9,34).Mutagenesis studies have been used to identify and characterize G␣ residues critical for guanine nucleotide binding (1,33,38,39), basal GDP release rates (34, 43), and GTP catalysis (19,43). O...

Identification and characterization of a potent and biologically-active PDE4/7 inhibitor via fission yeast-based assays

“…To identify BC54-resistant PDE4B2 alleles, we PCR amplified PDE4B2 under conditions previously-determined to introduce a modest level of mutations (see Materials and Methods). PCR using the FailSafe ™ PCR kit with buffers B, C, J or L and 20 cycles of amplification leads to a low percentage of mutant alleles, most of which carry a single amino acid change due to either a transition or transversion mutation ([38] and unpublished data). These PCR products were cloned by gap repair transformation into an expression vector plasmid, thus simultaneously inserting the PCR products into the vector and expressing the candidate proteins in a strain lacking PDE activity.…”

Sck1 Negatively Regulates Gpa2-Mediated Glucose Signaling in Schizosaccharomyces pombe

“…Crosses were performed on malt extract agar plates (MEA) containing 0.4% glucose (14), and tetrad dissection on YES plates was used for strain construction. Deletion and activated alleles of gpa2 ϩ (12,15), deletion, duplication, and kinase-dead alleles of sck1 ϩ (13, 16), a deletion allele of git3 ϩ (10), and the fbp1-lacZ reporter construct (17) have all been previously described.…”

“…However, if Sck1 is a negative regulator of Gpa2, the sck1 ϩ deletion would produce an elevated PKA phenotype. To enhance our ability to assess changes in Gpa2-mediated signaling, we examined these interactions in the context of a deletion of the git3 ϩ GPCR gene, as mutational activation of Gpa2 restores PKA activation in cells lacking Git3 (12). As seen in Fig.…”

“…Previously, we identified a large collection of mutationally activated alleles of the S. pombe gpa2 gene that allowed PKA activation to repress transcription of the fbp1 gene in cells lacking the Git3 GPCR, the Git5 G␤ subunit, or the G11 G␥ subunit (12). In this study, we set out to identify regulators of S. pombe Gpa2 G␣ by looking for proteins that preferentially bind mutationally activated Gpa2.…”

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

“…A widely used approach for precise allele replacement in fission yeast employs two sequential rounds of gene targeting [for recent examples see (Ahmed et al 2010; Ivey et al 2010; Singh et al 2010; Steiner et al 2011; Mudge et al 2012)]. This approach takes advantage of the fact that one can select positively or negatively for the ura4 + gene, which encodes orotidine-5′-phosphate decarboxylase (Bach 1987).…”