UC Riverside

Guanine nucleotide binding proteins (G proteins) and their cell surface G protein coupled receptors (GPCR) regulate many cellular processes in filamentous fungi, including growth, sexual, and asexual development. Neurospora crassa (N. crassa) has three G alpha subunits (GNA-1, GNA-2 and GNA-3), one G beta subunit (GNB-1), and one G gamma subunit (GNG-1). In this study, the three N. crassa G alpha subunits are analyzed for genetic epistasis with G beta subunit. Using double mutants lacking one G alpha gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each G alpha gene into the Δgnb-1 background genetic epistasis relationships were determined between G beta and G alpha subunit genes.

The length of the G protein signal is controlled by the duration of the GTP-bound alpha subunit; the intensity of the G protein signal is controlled by the level of activated G alpha subunits, which directly correlated with amount of protein and this regulation is not well known in filamentous fungi. Part of this dissertation focuses on investigation of the regulation of the G alpha subunits protein level, interaction with cytosolic proteins, translation, and degradation of G alpha subunit. Using endogenously tagged G alpha subunit (GNA-1), we identified cytosolic proteins associated with GNA-1. Putative GNA-1 interactors, which could affect stability of G alpha subunit, were checked for the level of the GNA-1 protein, including four 19S proteasome regulatory subunit deletion mutants. Our study shows that GNA-1 degraded by proteasome pathway. Level of G alpha subunit proteins in a whole cell extracts isolated from submerged liquid cultures are decreased in the Δgnb-1 and Δric8 strains relative to the wild type, with GNA-1 showing the greatest effect. Treatments with degradational inhibitors shows that protein turnover is not a mechanism responsible the low level of the G alpha subunit proteins in the Δgnb-1 and Δric8 backgrounds.

To determine if the translation is the regulatory mechanism of the G alpha proteins in the Δgnb-1 and Δric8 backgrounds, I stablished ribosomal profiling protocol for N. crassa. During adaptation of the protocol I found that cycloheximide pretreatment of living cells before harvesting and TRIzol extraction protocol are not suitable for N. crassa polysome-profiling protocol. I also found that GNB-1 and RIC8 protein associated with ribosomes or other elements of translational machinery.