cell wall biogenesis in neurospora crassa stephen free department of biological sciences suny...
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Cell wall biogenesis in Neurospora crassa
Stephen FreeDepartment of Biological Sciences
SUNY University at Buffalo
The Cell Wall is a critical organelle for the fungi.
• The cell wall protects the cell from desiccation, and other environmental stresses.
• The cell wall protects the fungus from other microorganisms.• The cell wall provides protection to fungal pathogen from
host defensives.• The cell wall components allow the cell to assess it’s
environment and act as the “upstream most” element for signal transduction pathways.
• The cell wall is necessary for polarized growth and morphology.
• Many antifungal agents target steps in cell wall biogenesis.
Key Points
• The N-linked galactomannan is used to cross-link proteins into the cell wall.
• DFG5 and DCW1 are cell wall α-1,6-mannanases/ mannantransferases which cross-link proteins into the cell wall.
• The CPS-1 polysaccharide synthase synthesizes a polysaccharide that is required for cross-linking proteins into the cell wall.
Cell Wall Polysaccharide Biogenesis
• Glucan synthase and chitin synthase are plasma membrane-associated, multi-pass transmembrane proteins.
• As they are synthesized, the linear glucan and chitin polymers are extruded into the cell wall space.
• Glucan and chitin are cross-linked to other polysaccharides and proteins in the cell wall matrix.
• The Gel1, Crh1, and Bgl2 glycosylhydrolase/ glycosyltransferase families are involved in creating a glucan/chitin matrix.
• Och-1 encodes a Golgi-associated α-1,6-mannosyltransferase.
• In N. crassa, OCH-1 adds the initial α-1,6-mannose of the galactomannan onto N-linked oligosaccharides.
OCH-1 is required for galactomannan synthesis
Galactomannan
Δoch-1 mutant characteristics:• Tight colonial
phenotype• Slow growth• Defective in aerial
hyphae• Abnormal conidiation
Microscopic examination:• Short, globular hyphae• Abnormal branching
pattern• Tight colonial growth
Δoch-1 has severe morphological defects
Western blot analysis of ACW-1 cell wall protein
• Δoch-1 is defective in incorporating cell wall proteins into the cell wall.
OCH-1 is required for protein cross-linking into the cell wall
WT secreted
Δoch-1
secreted
WT cell extract
Δoch-1 cell extract
WT secreted
Δoch-1 secreted
MNN-9 and KTR-1 catalyze galactomannan synthesis
MNN-9 functions to synthesize the α-1,6-mannose backbone and the KTR-1 functions to add the α-1,2-mannose side chains of the galactomannan.
Mnn-9
Ktr-1
MNN-9 and KTR-1 are needed for the effective incorporation of protein into the cell wall.
Western blot analysis of ACW-1 cell wall protein
Δoch-1 cell extractΔoch-1 secretedΔm
nn-9 cell extract Δm
nn-9 secreted
Δktr-1 secreted
WT
secreted
ΔKtr-1 cell extractW
T cell extract
Δmnn-9 is defective in incorporating ACW-1 into the wallΔktr-1 is less efficient than the wild type in incorporating ACW-1 into the cell wall
Identifying cross-linking enzymes that recognize the galactomannan
• α-1,6-mannanases are likely to cross-link glycoproteins to the glucan/chitin matrix.
• The N. crassa genome contains nine genes in the gh76 (α-1,6-mannanase) gene family and the single gene deletion library contains mutants for all nine genes.
• Deletion mutants for all nine genes were tested and two of them were identified as having a defect in cell wall biogenesis.
• Δdfg5, Δdcw1 double mutant resembles Δoch-1 phenotype.
DFG5 and DCW1 function to incorporate glycoproteins into the cell wall
• The Δdfg5, Δdcw1 double mutant is defective in incorporating cell wall proteins into the wall.
DFG5 and DCW1 function to incorporate glycoproteins into the cell wall
Western blot analysis of ACW-1 cell wall protein
WT secreted
Δdcw1 secreted
Δdfg5 secretedΔDcw
1,Δdfg5 secreted
• The DFG5 and DCW1 α-1,6-mannanases recognize the N-linked galactomannan, cleave the α-1,6-mannan backbone, and cross-link the N-linked galactomannan
into the glucan/chitin matrix
Conclusions
α-1,6-mannan backbone
Characterization of the Δcps-1 mutant
• The cps-1 gene encodes a 511 amino acid protein related to the capsid polysaccharide synthase from Crytococcus neoformans.
• In screening the single deletion library for female developmental mutants we noted that the Δcps-1 (polysaccharide synthase) mutant had a semi-colonial growth phenotype.
• Δcps-1 is sensitive to cell wall perturbation reagents.• RIP experiments demonstrate that the deletion of cps-1 is
responsible for the mutant phenotype.
CPS-1 is needed for the incorporation of cell wall proteins into the wall
M – Mol. Weight markers1. WT cell extract2. WT secreted protein3. Δcps-1 cell extract4. Δcps-1 secreted protein
M 1 2 3 4
Δcps-1 releases cell wall protein into the medium
1 2 3 4
1. Δcps-1 secreted protein2. Δcps-1 cell extract3. WT secreted protein4. WT cell extract
Western blot analysis of ACW-1 cell wall protein
CPS-1 is required for the incorporation of cell wall proteins into the cell wall.
Model for incorporating cell wall protein into the cell wall
• The CPS-1 generated polysaccharide is synthesized and extruded (reducing end first) into the cell wall space, and cross-linked into the glucan/chitin matrix.
• DFG5 and DCW1 binds to the reducing end of the CPS-1 polymer and places the reducing end of CPS-1 in its enzymatic active site.
• DFG5 and DCW1 then binds the N-linked galactomannan and uses the reducing end of cps-1 generated polysaccharide to attack its α-1,6-backbone.
• The terminal part of the galactomannan is released as a new glycosidic bond is generated between the cps-1 polysaccharide and the α-1,6-mannose backbone, which effectively cross-links the protein into the cell wall matrix.
Key Points
• The N-linked galactomannan is used to cross-link proteins into the cell wall.
• DFG5 and DCW1 are cell wall α-1,6-mannanases/ mannantransferases which cross-link proteins into the cell wall.
• The CPS-1 polysaccharide synthase synthesizes a polysaccharide that is used in cross-linking proteins into the cell wall.
Acknowledgements
Abhiram Maddi – och-1, dfg-5, and dcw-1Eleanor Sokolow – cps-1Ci Fu – cps-1
Funding by NIH grants R01 078589 and R03 AI103897 and the UB Foundation
α-1,3-glucan is a cell-type cell wall glucan
Wild type Δags-1
Δags-1 has a distinctive phenotype, affecting the production of conidia