Heterotrimeric (alpha-beta-gamma) G proteins are essential for many responses to environmental stimuli in eukaryotes. In the filamentous fungus Neurospora crassa, there are three known Galpha proteins (GNA-1 and GNA-3) and one Gbeta subunit (GNB-1). GNA-1 and GNA-2 have overlapping functions in regulating female fertility and vegetative growth. Only GNA-1 modulates GTP-dependent adenylyl cyclase activity (AC), while GNA-1 and GNA-2 both affect cAMP phosphodiesterase activity (PDE). GNA-3 regulates vegetative growth, but is not necessary for the sexual cycle. GNA-3 is implicated as a positive regulator of AC. These and other findings indicated that cAMP is important for vegetative, but not sexual functions. GNA-1 was the first microbial Galpha that could be classified in a mammalian family (Galpha/i). Systematic testing of deltagna-1 complementation by several mammalian Galpha genes was performed. The results also support separate signaling pathways for sexual and vegetative growth in N. crassa. Deltagnb-1 strains have greatly reduced GNA-1, but normal GNA-2 levels. It has been shown in other systems that beta-gamma regulates transcription. Beta-gamma proteins regulate phospholipase C (PLC) in mammals, leading to release of Ca/2+ from intracellular stores. The vacuole is the major Ca/2+ reservoir in N. crassa; and deltagnb-1 strains have vacuolar defects. Hence, GNB-1 may regulate gna-1 transcription and/or Ca/2+ mobilization, in N. crassa. Therefore, we hypothesize that G proteins differentially regulate 1) cAMP levels, 2) vacuolar function and 3) gene expression during vegetative and sexual development in Neurospora crassa. Our objectives are: 1) A direct interaction between GNA-1 and AC will be tested using reconstitution assays, co-immunoprecipitation, affinity chromatography and two-hybrid assays. Unknown targets of GNA-1 and GNA-2 will be identified by two- hybrid screens and genetic suppression analysis. Roles of GNA-1 and GNA-2 during the pheromone responses will be tested. 2) AC, PDE and cAMP levels will be measured in the mammalian Galpha transformants. Interactions between the mammalian Galpha's and GNB-1 will be tested in two-hybrid assays. Chimeras between complementing and non-complementing genes will be constructed to define regions important for vegetative or sexual functions. 3) AC and PDE activity will be measured to probe a role for GNA-3 in cAMP metabolism. Interactions between GNA-3 and other proteins will be determined using reconstitution, affinity chromatography and two- hybrid analysis. 4) Transcriptional versus post-translational regulation of GNA-1 levels by GNB-1 will be tested. AC, PDE and cAMP levels will be measured in deltagnb-1 strains. Vacuolar function will be assessed by measuring arginine and Ca/2+ pools and vacuolar ATPase activity PLC activity and Ca/2+ metabolism will be tested in deltagnb-1 mutants. Interactions between GNB-1 and other proteins will be defined using two- hybrid assays. These studies will elucidate G protein signaling pathways in filamentous fungi, speed the functional analysis of mammalian Galpha's and yield insight into G protein evolution. Furthermore, since homologues of N. crassa G alpha genes modulate virulence in several filamentous species, these investigations will also illuminate the role of G proteins in fungal pathogenesis, leading to new therapies for emerging fungal pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048626-09
Application #
6385786
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Anderson, Richard A
Project Start
1993-08-01
Project End
2001-11-30
Budget Start
2001-08-01
Budget End
2001-11-30
Support Year
9
Fiscal Year
2001
Total Cost
$31,132
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225