Figure 3.

Experimental organization and two representative results for GPA1-candidate GPCR interaction assessed by the split-ubiquitin system. (a) Schematic showing a simplified outline of the split-ubiquitin system assay: protein A is fused to the amino-terminal half of ubiquitin as an amino- or carboxy-terminal fusion (only the amino-terminal fusion orientation is shown here); protein B is fused to the carboxy-terminal half of ubiquitin, which in turn has a fused artificial transcription factor (PLV). Interaction of protein A with protein B brings the two halves of ubiquitin into close proximity and a functional ubiquitin molecule is restored. Ubiquitin specific proteases cleave off PLV, which translocates to the nucleus and activates transcription of target genes allowing for yeast growth. (b) Cartoon detailing the control (Nub_wt) and test (NubG) fusion protein construct orientations for sectors 1-4 in (c). (c) Schematic depicting the organization of the interaction assay plates in (d,e). The X represents the candidate GPCR open reading frame (ORF). Sectors 5-8 show the reciprocal assay. (d,e) Representative results for the ability of candidate GPCRs to interact with GPA1, the Gα subunit, on 1 mM methionine repression media. Diploid yeast containing GPA1 fusion constructs and either candidate Cand5 (d) or TOM1 (e) fusion constructs both grow on minimal media (not shown), but Cand5 specifically interacts with GPA1 and allows growth on the repression media (d, boxed sector) while TOM1 does not (e, boxed sector).