Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.In eukaryotic cells, a number of proteins are anchored to the outer leaflet of the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. The presence of the GPI anchor is thought to play an important role in the trafficking of these proteins and providing them with an attachment to the plasma membrane, and in the case of fungi, to the cell wall as well (24,41). Proteins destined to receive a GPI anchor are directed into the lumen of the endoplasmic reticulum (ER) by a typical signal peptide. The carboxyl termini of these proteins have a sequence motif that is recognized by a protein complex located in the ER, known as the GPI transamidase. The GPI transamidase complex cleaves the substrate protein at a position within this motif, termed the omega site, and transfers the GPI anchor en bloc to the newly generated C terminus of the protein.The structures of the GPI anchor in the trypanosome, yeast, and mammalian systems have been determined. Although there are differences in the various substituents present on the GPI anchors produced by these organisms, all GPI anchors appear to share a common core structure (15,19,20). This core structure consists of a phosphatidylinositide (or inositolcontaining sphingolipid) with an attached oligosaccharide chain that is terminated with a phosphoethanolamine residue. The linkages between the sugar units within the carbohydrate chain are conserved, and the amino group of the phosphoethanolamine moiety is used to attach the GPI anchor to the C terminus of the target protein. The organization of this basic GPI anchor structure is as follows: protein-phosphoethanolamine-6Mannose␣1-2Mannose␣1-6Mannose␣1-4Glucos-amine␣1-6inositol-phospholipid.The process of GPI anchor production and attachment is mediated by the concerted actions of approximately 20 proteins, which are organized into biosynthetic complexes in the ER membrane. Seven primary steps ha...
Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.
Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.
The glycosylphosphatidylinositol (GPI) transamidase contains five known subunits and functions in the lumen of the ER to produce GPI-anchored proteins. The transamidase cleaves proteins containing a GPI anchor attachment signal at their C terminus and generates an amide bond between the newly generated carboxyl terminus of the protein and a GPI anchor. We have identified and characterized GPIT-1 and GPIT-2, two of the transamidase subunits from Neurospora crassa. GPIT-1 and GPIT-2 are homologs of the human PIG-T and PIG-U transamidase subunits respectively. We demonstrated that GPIT-2 is required for the addition of GPI anchors onto GPI-anchored proteins. We employed the Neurospora RIP (repeat-induced point mutation) phenomenon to generate 106 "noncritical" amino acid changes in GPIT-1 and 84 "noncritical" amino acid changes in GPIT-2. We used the data to evaluate three-dimensional models for the structures of GPIT-1 and GPIT-2. The mutational data for GPIT-1 is consistent with a multiple-blade propeller structure containing a central channel. The mutational analysis for GPIT-2 supports a structural model based on the karyopherin alpha subunit.
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