Two-hybrid system: yeast GAL4-BD/GAL4-AD

In the Frq2 screen, 20 positive clones were identified corresponding to ric8a. A similar screen using Frq1 as bait recovered no positive interactors, even though Frq1 and Frq2 are 95% identical to each other, and no interaction between Frq1 and ric8a was observed in two hybrid assays.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from embryo cDNA expression library).

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced as a recombinant fusion protein in bacterial system.

ric8a pulled down Frq2 but not Frq1.

ric8a binds Frq2 but not to Frq1.

Source was cell extract of HEK293 cell line; bait produced from transfected construct; prey produced from transfected construct.

The Frq2 residues R94 and T138 are important in binding to ric8a, since introduction of either of these residues into Frq1 (K94R or S138T) confers ric8a binding activity.

Source was cell extract of HEK293 cell line; bait produced from transfected construct; prey produced from transfected construct.

The Frq2-ric8a interaction is inhibited by the small molecule FD44.