Positive Regulation of Adenylyl Cyclase Activity by a Galphai Homolog in Neurospora Crassa

Fungal Genet Biol. 1999 Feb;26(1):48-61. doi: 10.1006/fgbi.1998.1101.

Abstract

GNA-1 and GNA-2 are two G protein alpha subunits from the filamentous fungus Neurospora crassa. Loss of gna-1 leads to multiple phenotypes, while Deltagna-2 strains do not exhibit visible defects. However, Deltagna-1Deltagna-2 mutants are more affected in Deltagna-1 phenotypes. Here we report a biochemical investigation of the roles of GNA-1 and GNA-2 in cAMP metabolism. Assays of Mg2+ ATP-dependent adenylyl cyclase activity (+/-GppNHp) in extracts from submerged cultures indicated that Deltagna-2 strains were normal, whereas Deltagna-1 and Deltagna-1Deltagna-2 strains had only 10-15% the activity of the wild-type control. Levels of the Gbeta protein, GNB-1, were normal in Deltagna-1 strains, excluding altered GNB-1 production as a factor in loss of adenylyl cyclase activity. Steady-state cAMP levels in Deltagna-1 and Deltagna-1Deltagna-2 mutants were reduced relative to wild-type under conditions that result in morphological abnormalities (solid medium), while levels in submerged culture were normal. cAMP phosphodiesterase activities in submerged cultures of Deltagna-1 and/or Deltagna-2 strains were lower than in wild-type; the individual deletions were additive in decreasing activity. These results suggest that in submerged culture, N. crassa, like mammalian systems, possesses compensatory mechanisms that maintain cAMP at relatively constant levels. Furthermore, the finding that Mg2+ATP-dependent adenylyl cyclase activity in wild-type cell extracts could be inhibited using anti-GNA-1 IgG suggests that GNA-1 directly interacts with adenylyl cyclase in N. crassa.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / metabolism
  • Acetyltransferases*
  • Adenosine Triphosphate / metabolism
  • Adenylyl Cyclase Inhibitors
  • Adenylyl Cyclases / genetics
  • Adenylyl Cyclases / metabolism*
  • Animals
  • Antibodies, Fungal / pharmacology
  • Antibody Specificity
  • Fungal Proteins / antagonists & inhibitors
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / physiology*
  • Glucosamine 6-Phosphate N-Acetyltransferase
  • Immunoglobulin G / pharmacology
  • Mutation
  • Neurospora crassa / enzymology*
  • Neurospora crassa / genetics
  • Neurospora crassa / growth & development
  • Rabbits
  • Radioimmunoassay

Substances

  • Adenylyl Cyclase Inhibitors
  • Antibodies, Fungal
  • Fungal Proteins
  • Immunoglobulin G
  • Adenosine Triphosphate
  • Acetyltransferases
  • Glucosamine 6-Phosphate N-Acetyltransferase
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Adenylyl Cyclases