Figure 4. Defining the minimal ankyrin-binding site on InsP₃ receptor N-terminus.

A, Ankyrin-B membrane binding domain (bait) and a series of InsP₃ receptor constructs (prey, see Figure 3) were co-transformed into AH109 yeast to identify a minimal region of the InsP₃ receptor responsible for interaction with ankyrin-B. Co-transformants were selected on –LT media and analyzed for HIS3 reporter gene activation by evaluating growth on –AHLT after five days incubation. Positive interaction is seen with all constructs, with the smallest interacting region of the InsP₃ receptor corresponding to amino acids 924–991. Note that this region is located on the cytoplasmic N-terminus of the InsP₃ receptor. B, InsP₃ receptor lacking residues 978–1582 fails to interact with ankyrin-B membrane-binding domain in yeast. Positive control for experiments was InsP₃ receptor 924–1582. Positive interaction was assessed after 5 days on AHLT medium.