Activation of Neurospora crassa soluble adenylate cyclase by calmodulin.
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ABSTRACT:
SUBMITTER: Reig JA
PROVIDER: S-EPMC1144071 | biostudies-other | 1984 Jul
REPOSITORIES: biostudies-other
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Project description:A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.
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Project description:The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.
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Project description:We have identified a new gene encoding the G protein alpha subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Galpha proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the Deltagna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to Deltagna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Deltagna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Deltagna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of Deltagna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Deltagna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.
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