Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium.
As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator 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 centrifugation 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 activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.
Lilly
P J
PJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Devreotes
P N
PN
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins
D005656:Fungal Proteins; D006150:Guanine Nucleotides; D015800:Protozoan Proteins; C089420:dagA protein, Dictyostelium; D000262:Adenylyl Cyclases