Article Figures & Data
Figures
- 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+ G418r 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.
Tables
- Table 1.
Strain list
Strain Genotype CHP468 h − ade6-M216 his7-32 ura4::fbp1-lacZ fbp1::ura4+gpa2Δ::ura4− SLP29 h − ade6-M216 his7-366 leu1-32 ura4::fbp1-lacZ fbp1::ura4+git5Δ::his7+git11Δ::kan gpa2Δ::ura4− DIP10 h − his7-366 leu1-32 ura4::fbp1-lacZ fbp1::ura4+git3Δ::kan gpa2Δ::ura4− MZP2 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2Δ::ura4+ MZP5 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(R176H) MZP6 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(F62S) MZP7 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(K270E) MZP8 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(L57P) MZP9 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(K270N) MZP10 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(S149F) MZP12 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(T325A) MZP13 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(V90A) MZP14 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(V237A) MZP15 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(T49A) MZP18 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2+ FYP1 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(I56S) FYP2 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(S81F) FYP3 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(E184D) - Table 3.
Bypass of git3Δ defect in glucose signaling by activated gpa2 allelesa
gpa2 allele Domain β-Gal activity gpa2Δ 1,240 ± 166 gpa2 + 390 ± 101 gpa2(I56S) Linker 1 15 ± 13 gpa2(L57P) Linker 1 7 ± 1 gpa2(F62S) Linker 1 9 ± 3 gpa2(S81F) Helical 13 ± 8 gpa2(V90A) Helical 8 ± 3 gpa2(S149F) Helical 7 ± 1 gpa2(T49A) GTPase 8 ± 1 gpa2(R176H) GTPase 5 ± 0 gpa2(E184D) GTPase 658 ± 90 gpa2(K270E) GTPase 7 ± 1 gpa2(K270N) GTPase 5 ± 3 gpa2(T325A) GTPase 37 ± 20 gpa2(V327A) GTPase 11 ± 4 ↵a β-Galactosidase activity was determined from three or four independent cultures grown overnight in yeast extract rich medium containing 8% glucose to a final density of ∼1 × 107 cells/ml. The assays were carried out as previously described (30). All strains carry a deletion of the git3+ GPCR gene, which can be suppressed by mutational activation of Gpa2 to restore repression of fbp1-lacZ expression. The values represent the mean specific activities per milligram of soluble protein ± standard errors.
