The Role of Cross Pathway Control-2 (cpc-2) in Filamentous Fungus Neurospora Crassa

In the filamentous fungus Neurospora crassa, heterotrimeric G protein pathways are major signaling cascades through which the fungus senses and adapts to its environment. The characterized Gβ subunit of N. crassa, GNB-1, has seven tryptophan-aspartate (WD) repeats, predicted to result in a β propeller structure. Another related N. crassa protein, called Cross Pathway Control-2 (CPC-2), also has a seven WD repeat structure and possesses 70% positional identity with Receptor for Activated C Kinase-1(RACK-1), a multifaceted scaffolding protein in mammals. Accumulating evidence in many organisms shows that RACK1 homologs can act as Gβ subunits. Previous work demonstrated that CPC-2 plays an important role during general amino acid control in N. crassa, along with having a role in overall growth and female fertility. My research investigated a possible role for cpc-2 in the G protein signaling pathway, and also investigated genetic epistasis between cpc-2, gnb-1 and the Gα genes in N. crassa.\tIn Chapter 2, genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3Q208L allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. In Chapter 3, genetic epistasis genes showed that gna-3 is epistatic to cpc-2 during conidiation in submerged culture. gna-1 exhibited partial epistasis to cpc-2 during submerged culture conidiation. gna-3, gnb-1 and gng-1 operate downstream of cpc-2 during aerial hyphae height development. Apical extension assays showed that cpc-2 is epistatic to gnb-1 and gng-1. Similar to the results for gnb-1 in Chapter 2, none of the activated Gα alleles restored fertility to the sterile Δcpc-2 mutant. Analysis of apical extension rates on medium supplemented with -amino triazole (3AT) revealed a previously unknown role for gna-1 and gna-3 in general amino acid control. Yeast two hybrid mating assays revealed that CPC-2 interacts with GNA-1 and GNA-3.