Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of alpha beta and gamma subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the G-alpha subunit. Once activated, G-alpha-GTP dissociates from the beta gamma dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes incellular metabolism and physiology. Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three G-alpha (GNA-1, GNA-2, GNA-3) and one G-beta (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. GNA-1 was the first identified microbial G-alpha that belongs to a mammalian superfamily (G-alphai). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, delta-gna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the delta-gna-1 mutant is male fertile but female sterile. Biochemical studies have shown that delta-gna-1 strains have lower adenosine 3′-5′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian G-alphai superfamily members were transformed into a delta-gna-1 strain, and complementation of delta-gna-1 defects investigated. G-alphas, which is not a member of G-alphai superfamily was used as a control. These mammalian G-alpha genes were able to rescue the vegetative growth rate defect of the delta-gna-1 strain in the following order: G-alphaz>G-alphao>G-alphas>G-alphat>G-alphai. In contrast, only G-alphao was able to complement the sexual defect of a delta-gna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian G-alpha genes restored the sensitivity of heat treatment to a wild type level. These results from mammalian G-alpha gene complementation of a delta-gna-1 strain study suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. GNB-1, a G protein beta subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 shares 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gbeta genes (~66%). A delta-gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated.
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Heterotrimeric G Protein-Mediated Signal Transduction in Neurospora crassa