Abstract

In previous work, the Dictyostelium discoideum gene encoding the cAMP-binding protein CABP1 was isolated and cloned. Subsequent studies revealed that certain polypeptides are related in their amino acid sequence to CABP1. Each of these polypeptides possesses a region of high glycine, tyrosine, proline, glutamine, and alanine (GYPAQ) content which forms the basis for their similarity. In the present study, the genes encoding seven of these related D. discoideum polypeptides were cloned into the two-hybrid system vectors and subjected to the yeast two-hybrid assay. The p34, p31, CABP1A, CABP1B, and 6C proteins each homodimerized in the assay. Furthermore, interactions were observed between the following pairs of proteins: p34 and p31, CABP1A and CABP1B, CABP1A and 6C, CABP1B and 6C, p34 and 6C, p31 and 6C, and p24 and 6C. Some of the interactions were also verified using a blot overlay assay. Dictyostelium annexin did not interact with any of the other related proteins, nor did it homodimerize. To determine whether the GYPAQ-rich regions are mediating the interactions, portions of the gene encoding the 6C protein were isolated and cloned into the two-hybrid vectors and the assay was performed again. Genetically deleting the GYPAQ-rich region of 6C had the effect of abolishing or diminishing all the interactions that were observed with the intact 6C. Conversely, the GYPAQ-rich region behaved like the intact 6C. The GYPAQ-rich region therefore appears to be playing a role in the mediation of interactions with 6C