Heterotrimeric (a??) G proteins are essential components of signal transduction pathways that regulate environmental sensing, growth and development in eukaryotes. G protein coupled receptors (GPCRs) have been characterized as guanine nucleotide exchange factors (GEFs) for Ga subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as the RIC8 protein, has emerged as an important mechanism for Ga regulation in animals. RIC8 is required for asymmetric cell division in both Caenorhabditis elegans and Drosophila. Loss of ric8 leads to reduced levels of G proteins in Drosophila, suggesting a chaperone function in addition to GEF activity. RIC8 is absent from plants, protists and the yeast Saccharomyces cerevisiae. There are striking similarities between the functions of RIC8 in animals and the model eukaryotic filamentous fungus Neurospora crassa. Deletion of ric8 leads to severe defects in growth and asexual and sexual development in Neurospora, similar to those observed for a mutant lacking the Ga genes gna-1 and gna-3. ?ric8 strains have greatly reduced levels of G proteins, constitutively-activated alleles of gna-1 and gna- 3 rescue many defects of ?ric8 mutants and preliminary results support a physical interaction between RIC8 and GNA-1 and GNA-3. Furthermore, filamentous fungi are excellent models for asymmetric cell division in animals and Neurospora is the best-characterized microbial system that contains a RIC8 homologue. Based on these observations, we hypothesize that RIC8 modulates cell division in Neurospora by acting as a GEF for GNA-1 and GNA-3 and/or by serving as a protein chaperone that protects the G protein heterotrimer from degradation by the proteasome.
The Specific Aims are: 1) Determine the mechanism(s) that lead to low levels of G proteins in strains lacking the G?? dimer or RIC8. The stability of G protein subunits in ?ric8 and G?? mutants will be determined in the presence of agents that influence the proteolytic activity of vacuolar proteases or the proteasome. G protein levels will be measured in mutants lacking components of E3 ligase complexes. 2) Characterize interactions between RIC8 and G proteins. Mutants lacking ric8 and G?? genes will be analyzed. Physical interactions between RIC8 and G proteins will be probed using co-immunoprecipitation. RIC8 will be tested for Ga GEF activity. 3) Explore the possibility that RIC8 acts in concert with Ga proteins to regulate septation and asymmetrical cell division. The localization of actin, tubulin and GFP-tagged RIC8, RHO-4, Ga and G? proteins will be determined in various mutant strain backgrounds. RIC8 will be tested for GEF activity toward RHO-4. 4) Identify novel pathway components and bypass mechanisms using genetic screens. Mutations in genes that influence ric8 function in animals will be tested for effects in the ?ric8 background. A new Single Nucleotide Polymorphism Cleaved Amplified Polymorphic Sequence-based method (SNP-CAPS) will be used to clone suppressors isolated in the ?gna-1 ?gna-3 and ?ric8 genetic backgrounds. Mutants from the knockout project will be screened for defects common to ?ric8 and G protein mutants.
These studies will reveal novel G protein signaling mechanisms in Neurospora crassa that are shared with animals, but are not found in the yeast Saccharomyces cerevisiae. The information obtained will be applicable to development of new drugs that target RIC8-G protein pathways in human cells. Moreover, because RIC8 is conserved in filamentous fungi and G proteins are required for virulence in this class of organisms, the studies will also reveal new targets for antifungal agents that can be used to treat infections in humans.
