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Regulation of Heterotrimeric G Alpha Subunits in the Filamentous Fungus, Neurospora crassa

  • Author(s): Wright, Sara Josephine
  • Advisor(s): Borkovich, Katherine A
  • et al.
Abstract

Heterotrimeric G protein signaling pathways are crucial for eukaryotic cells to regulate environmental sensing, growth and development. G protein signaling involves a membrane spanning receptor called a G protein-coupled receptor (GPCR) that can be activated by an extracellular signal. GPCR's are bound intracellularly by a trimer of proteins: the Gα subunit, which binds and hydrolyzes GTP, and a tightly associated Gβγ dimer. Upon receptor activation, a conformational change causes the Gα subunit to exchange GDP for GTP and become dissociated from the GPCR and Gα. The GPCR is thus considered a guanine-nucleotide exchange factor (GEF) for the Gα subunit. The Gα subunit has intrinsic GTPase activity which converts GTP to GDP, causing Gα to re-associate with the receptor and Gβγ.

This dissertation focuses on the regulation of G protein signaling in the filamentous fungus Neurospora crassa. N. crassa has three Gα proteins (GNA-1, GNA-2, GNA-3), one Gβ (GNB-1), one Gγ (GNG-1), and 10 predicted GPCR's. The Gα subunit GNA-1 in Neurospora crassa has been shown to be vital for proper cell growth and differentiation, and a variety of functions have been identified for GNA-1. Using a yeast two-hybrid screen, I identified interesting potential partners of GNA-1 including two metabolic enzymes, two proteins involved in protein synthesis, an actin nucleating protein, and proteins involved in vitamin synthesis, lipid, and two fungal specific proteins with unknown function.

The majority of this dissertation focuses on a homolog of a non-GPCR GEF, RIC8, which has been shown to be important in mechanisms for Gα regulation in animals. RIC8 is present in filamentous fungi, but is absent from the genomes of Saccharomyces cerevisiae and sequenced protists, including Dictyostelium discoideum. Deletion of ric8 leads to defects in polar growth and asexual and sexual development in Neurospora, and constitutively activated alleles of gna-1 and gna-3 rescue many defects of δric8 mutants. RIC8 interacts in vitro with GNA-1 and GNA-3 and preliminary results show that RIC8 has GEF activity toward GNA-1. This data shows both a functional and physical interaction between RIC8, GNA-1 and GNA-3. Hyphae from Neurospora δric8 mutants have small cell compartments that contain nuclei that are smaller and morphologically abnormal compared to wild-type, which also suggest a role in polar cell growth. Taken together, our results support a role for RIC8 and G proteins in regulation of polar growth in Neurospora.

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