Characterization of the Neurospora crassa cell wall and glycosylphosphatidylinositol (GPI)-anchor biosynthetic pathways
Bowman, Shaun Matthew
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The cell wall plays a vital role in the physiology of the filamentous fungus Neurospora crassa , but its synthesis, structure, and remodeling dynamics have not been well defined. To this end, N. crassa mutants affected in two biosynthetic pathways involved in cell wall formation, protein mannosylation and glycosylphosphatidylinositol (GPI)-anchor addition, have been isolated and characterized. These pathways are important for the production of cell wall glycoproteins, which are involved in the synthesis, assembly, organization, and remodeling of the cell wall. The mutants exhibit reduced rates of growth, altered hyphal and gross colony morphologies, and pronounced cell wall defects. In addition, these cell wall mutants are unable to produce many of the characteristic cell types formed as part of the normal N. crassa life cycle. In chapter 2, a novel genetic mapping and PCR-based sequencing assay was used to the clone the mnt-1 gene, which encodes an alpha-1,2-mannosyltransferase. The mnt-1 gene was shown to be required for the synthesis of the galactomannan component present on glycoproteins in the cell wall. Mnt-1 mutants have altered cell wall carbohydrate composition and are unable to repress the onset of the asexual developmental program. The work in chapter 3 details the identification of the gpip-1, gpip-2, gpip-3 , and gpit-1 genes, which are involved in the addition of the GPI-anchor to select cell wall glycoproteins. The gpip-1, gpip-2 , and gpip-3 genes encode phosphoethanolamine transferases that function in the addition of phosphoethanolamine groups to the GPI-anchor during anchor biogenesis. The gpit-1 gene encodes a component of the GPI transamidase complex involved in the transfer of the completed anchor structure to the target protein. The GPI-anchor mutants experience a significant degree of cell lysis, have altered cell wall carbohydrate and protein compositions, and are deficient in the production of a number of putative GPI-anchored cell wall proteins. The characterization of the protein component in the mutant and wild-type cell walls allowed for the identification of several GPI-anchored and non-anchored cell wall proteins. Overall, the results of this doctoral work demonstrate that the addition of galactomannan to cell wall proteins is an important element of cell wall biogenesis. The work also highlights the significance of the GPI-anchor in directing GPI-anchored proteins to the cell wall and the importance of those proteins for cell wall biosynthesis and remodeling. The addition of galactomannans and GPI-anchors are required for the normal morphology and development of N. crassa .