Biomass Depolymerization projects
Characterizing the Cellulase Secretion Pathway in Filamentous Fungi
Filamentous fungi secrete cellulases that act to degrade complex plant cell walls into simple sugars that the fungal cell can use for growth. In order to be secreted the cellulases must pass through an ordered array of intracellular organelles, which are collectively referred to as the secretory pathway. We are using Neurospora crassa, as a model filamentous fungus, to study components that are required for transport of cellulases through the endoplasmic reticulum, the first organelle of the secretory pathway. Our hope is to find targets for genetic manipulation that might lead to the design and engineering of filamentous fungal strains with enhanced secretion capability.
This project uses reverse genetic and cell biological approaches to reveal and characterize key components required for efficient trafficking of cellulase enzymes through the secretory pathway of the filamentous fungus Neurospora crassa. We are primarily focusing on understanding the role of cargo adaptors in the efficient exit of these enzymes from the first compartment of the secretory pathway, the endoplasmic reticulum (ER). Using the techniques of fluorescent protein tagging, reverse genetics, trafficking kinetic analysis, biochemistry and transcriptional expression analysis, we have discovered that the two most abundantly secreted cellulases of N. crassa, CBH-1 and CBH-2, rely on distinct ER cargo adaptors for their efficient ER exit.
An important mechanism to ensure efficient secretion of proteins is selective enrichment of cargo proteins into budding transport vesicles at the endoplasmic reticulum (ER), followed by delivery of the resulting vesicles to the Golgi apparatus. This process is mediated by a number of different proteins that are localized within the ER membrane as well as in the cytosol. We are interested in identifying key players in this process during cellulase secretion from the filamentous fungus Neurospora crassa. To achieve this goal, we are currently investigating a set of mutants that are defective for cellulase secretion and that lack a specific class of ER-resident proteins. The project will focus on the characterization of these proteins in mediating the exit of cellulases from the ER. We are developing assays to assess cellulase localization in these mutants, as well as any ER-exit defects that may be unique to specific cellulases. These assays will permit testing for cellulase secretion defects in a selection of additional candidate mutants, which lack other ER-resident proteins that are either up-regulated when N. crassa is grown cellulose or have functions that are similar to the mutants that are already under investigation. The protein trafficking defect elicited by the absence of these ER-resident proteins has the potential to cause ER stress, with subsequent induction of ER stress response pathways. Therefore, we are additionally testing these mutants for ER stress response pathway induction and will determine how such pathways influence cellulase secretion.
Published in 2014
Plant Cell Wall-Degrading Enzymes and Their Secretion in Plant-Pathogenic Fungi, C. P. Kubicek, T. L. Starr, N. L. Glass, Annual Review of Phytopathology, 52, pp. 427-451, June 16, 2014.