Investigating the mechanism of enzyme translocation through the secretory pathway of Neurospora crassa

The saprotrophic fungus, Neurospora crassa has evolved to be exceptionally good at secreting a large concentration of enzymes necessary to degrade complex carbohydrates. It is well established that N. crassa transcriptionally regulates these enzymes in order to efficiently utilize plant cell wall components as a carbon source. However, downstream regulation of the secretory pathway likely improves the efficiency of plant cell wall degradation and the mechanisms through which this occurs are still obscure. To elucidate these unknown mechanisms, we conducted a forward genetic screen to identify and characterize defective trafficking mutants that cannot translocate cellulases in the ER or that improperly retain cellulases in the ER instead of secreting them into the extracellular environment.. We generated a mutant library using random mutagenesis of a strain with a GFP tagged endoglucanase (EG-2), to screen for mutants with mis-localized EG-2 via microscopy. From the screen, we identified a particularly interesting mutant (10C2) that has EG-2-GFP mislocalization to the ER, a growth defect and temperature sensitivity. We used bulked segregant analysis to identify the putative causal mutation. The potential gene of interest is a scaffolding protein that interacts with multiple different proteins in various organisms, thus exhibiting diverse cellular functions in different organisms. However, its function in N. crassa has not been explored, and preliminary data suggests that this mutation causes a general secretion defect, as opposed to one specific to the cellulolytic response. We plan to assess its function by examining its localization in the cell and interacting partners and its role in ER to Golgi trafficking of cellular proteins and cellulases. Identifying unknown components that play a role in secretion in filamentous fungi, using cellulase trafficking as a tool will provide a better understanding of the regulation of the secretory pathway in filamentous fungi.