Functional dissection of adenylate cyclase R, an inducer of spore encapsulation.
Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr(-) cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr(-) phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr(-) phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain.
College of Life Sciences, University of Dundee, Dundee DD15EH, Scotland, United Kingdom.
Zhi-hui
ZH
Chen
Christina
C
Schilde
Pauline
P
Schaap
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D004023:Dictyostelium / Q000201:enzymology*; D019281:Dimerization; D005799:Genes, Dominant; D000071677:Histidine Kinase; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011494:Protein Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013170:Spores
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D011494:Protein Kinases; D000071677:Histidine Kinase; D000262:Adenylyl Cyclases