Activated alleles of the Schizosaccharomyces pombe gpa2+ Galpha 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 Galpha 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 Gbetagamma 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 Galpha function, thus extending our understanding of Galpha protein structure-function relationships.

Fig. 1.

Coexpression of the Git5 Gβ enhances a two-hybrid interaction between the Git3 GPCR and the Gpa2 Gα subunit. (A) Strain YRG2 was transformed to Leu⁺ with a prey plasmid that expressed the carboxy-terminal half of Git3 beginning with the third cytoplasmic loop. This transformant was transformed to Trp⁺ G418^r with bait and facilitating plasmids. The bait plasmids expressed either wild-type Gpa2⁺ or mutationally activated Gpa2(R176H). The facilitating plasmids expressed either the Git5 Gβ or the lambda cI repressor protein (−Git5 Gβ, as a negative control). Three independent transformants were replica plated onto SC-Leu, Trp, His with or without 7.5 mM 3AT and grown for 3 days before being photographed. (B) Growth of transformants on SC-Leu, Trp, His. (C) Growth of transformants on SC-Leu, Trp, His containing 7.5 mM 3AT. (D) X-Gal filter lift of transformants to detect β-galactosidase expression that resulted from the protein-protein interaction.

Fig. 2.

Determination of the level of Gpa2 expression required to distinguish between wild-type and mutationally activated proteins. Strains CHP468 (gpa2Δ) and SLP29 (gpa2Δ git5Δ git11Δ) were transformed to Leu⁺ with pNMT41-1 (empty vector) (columns A), pDI2 (pNMT41-gpa2⁺) (columns B), pDI53 [pNMT41-gpa2(R176H)] (columns C), or pDI28 (pNMT41-myc-gpa2⁺) (columns D). Transformants were pregrown on PM-Leu medium with and without 10 μg/ml thiamine before being replica plated onto 5-FOA medium. The plates were incubated for 2 days at 30°C before being photographed.

Fig. 3.

Crystal structure of transducin Gα bound to GDP, showing the locations of Gpa2 residues altered by activating mutations. The structure (Protein Data Base [PDB] ID 1TAG) was generated using RasMol. GDP is displayed in white using the spacefill format. (A and B) The 5 residues (T49 [blue], S149 [cyan], K270 [red], T325 [yellow], and V327 [green]) that contact the guanine nucleotide are displayed in the spacefill format. Two orientations of the structure are provided to show the contacts with GDP. (C) The 3 residues (I56 [blue], L57 [green], and F62 [red]) located in the linker 1 region are displayed in the spacefill format. (D) The 2 residues (S81 [blue] and V90 [green]) located in the αA helix of the helical domain are displayed in the spacefill format.

Fig. 4.

Alignment of Gα subunits and S. pombe Rheb protein, together with identification of altered residues in activated S. pombe Gpa2 proteins. S. pombe (Sp) Gpa2 (CAB16244), S. cerevisiae (Sc) Gpa2 (NP_010937), human transducin α subunit (NP_000163), S. pombe Gpa1 (CAA21150; Gα of the pheromone pathway), and S. pombe Rhb1 (CAA22291) were aligned using Clustal W (44) and displayed using BOXSHADE 3.21. Identical residues are shaded in black. Conserved residues are shaded in gray. Dashes were introduced by the alignment software. The locations of altered residues that activate S. pombe Gpa2 (Table 3) are indicated above the alignment, while the locations of two altered residues that activate S. pombe Rhb1 (45) are indicated below the alignment.