Skip to main content
. 2006 Aug;5(8):1287–1300. doi: 10.1128/EC.00109-06

FIG. 4.

FIG. 4.

GPR-4 is coupled to GNA-1. (A) Levels of heterotrimeric G protein subunits. A cell fraction enriched for plasma membranes was isolated from VM-glycerol and VM-sucrose plate cultures, and samples containing 30 μg protein were analyzed by Western blot using G protein antisera (see Materials and Methods for details). Strains are 74A (wild type) and 7-32 (Δgpr-4 mutant). The asterisk denotes a nonspecific band recognized by the GNA-1 antiserum. (B) Epistasis analyses between gpr-4 and the three Gα genes. Experimental conditions and dry mass determination are the same as for Fig. 3A. Strains are 74A (wild type), 7-32 (Δgpr-4), 1B8 (Δgna-1), a1r54 (Δgna-1 Δgpr-4), a29-1 (Δgna-2), a2r2 (Δgna-2 Δgpr-4), 2a1 (Δgna-3), a3r1 (Δgna-1 Δgpr-4), Rmi2 (Δgpr-4 gpr-4+), Δ1gna-1Q204L (Δgna-1 gna-1Q204L), and a1rQ204L-25 (Δgpr-4 Δgna-1 gna-1Q204L). Data are the averages of results from two representative independent experiments, and errors are calculated as standard errors. (C) Yeast two-hybrid assay. Left panel: β-galactosidase assay. Yeast transformants carrying the indicated ORFs or domains in the pGBKT7 (left of slash) or pGAD424 (right of slash) vectors were cultured on SD minus leucine and tryptophan plates for ∼2 days. β-Galactosidase activity was determined using filter assays (see Materials and Methods for details), and photos were taken 2 h after application of the 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside substrate solution. Right panel: growth assay. Serial dilutions were made of SD minus leucine and tryptophan liquid cultures of yeast strains with the indicated ORFs or protein domains. Aliquots corresponding to each concentration of cells (107, 106, or 105 cells/ml) were spotted onto SD plates lacking adenine, histidine, leucine, and tryptophan, supplemented with 5 mM 3-aminotriazole. The plate was photographed after incubation at 30°C for 3 days. GNA-1, GNA-1 ORF; GNA-2, GNA-2 ORF; GNA-3, GNA-3 ORF; CT, carboxy terminus of GPR-4; +/+, positive control containing pGBKT7-53 and pGADT7-Rec (Clontech); −, pGBKT7 (left) or pGAD424 (right) empty vector.