IDENTIFICATION OF CRAC, A CYTOSOLIC REGULATOR REQUIRED FOR GUANINE-NUCLEOTIDE STIMULATION OF ADENYLYL-CYCLASE IN DICTYOSTELIUM

As previously reported, guanine nucleotide regulation of adenylyl cycl ase activity in the Dictyostelium mutant synag 7 can be restored in vi tro by addition of a high speed supernatant prepared from wild-type ce lls (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 151 21-15125). We have designated this activity CRAC, for cytosolic regula tor of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient ce ntrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activ ity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immu noblots of fractions from each purification step show that activity an d the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a po rtion of the deduced N terminus of a gene, dagA, isolated from a mutan t similar to synag 7. Expression of the dagA cDNA in synag 7 cells res tores both the 88 kDa band and CRAC activity.