Dr. Steve Free - Department of Biological Sciences

Report
Cell wall biogenesis in Neurospora crassa
Stephen Free
Department 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.
Fungal Cell Wall Structure
Key Points
• The N-linked galactomannan is used to crosslink proteins into the cell wall.
• DFG5 and DCW1 are cell wall α-1,6mannanases/ mannantransferases which
cross-link proteins into the cell wall.
• The CPS-1 polysaccharide synthase synthesizes
a polysaccharide that is required for crosslinking 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.
Cell Wall Protein Biosynthesis
OCH-1 is required for galactomannan synthesis
• Och-1 encodes a Golgiassociated α-1,6mannosyltransferase.
• In N. crassa, OCH-1
adds the initial α-1,6mannose of the
galactomannan onto
N-linked
oligosaccharides.
Galactomannan
Δoch-1 has severe morphological defects
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
OCH-1 is required for protein cross-linking
into the cell wall
Western blot analysis of ACW-1 cell wall protein
• Δoch-1 is defective in incorporating cell wall proteins into the
cell wall.
MNN-9 and KTR-1 catalyze galactomannan
synthesis
MNN-9 functions to
synthesize the α-1,6mannose backbone and
the KTR-1 functions to add Ktr-1
the α-1,2-mannose side
chains of the
galactomannan.
Mnn-9
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
Δmnn-9 is defective in incorporating ACW-1 into the wall
Δktr-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-1,6mannanase) 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.
DFG5 and DCW1 function to incorporate glycoproteins into the
cell wall
• Δdfg5, Δdcw1
double mutant
resembles Δoch-1
phenotype.
DFG5 and DCW1 function to incorporate glycoproteins into the
cell wall
Western blot analysis of ACW-1 cell wall protein
• The Δdfg5, Δdcw1 double mutant is defective in
incorporating cell wall proteins into the wall.
Conclusions
• The DFG5 and DCW1 α-1,6mannanases recognize the
N-linked galactomannan,
cleave the α-1,6-mannan
backbone, and cross-link
the N-linked galactomannan
into the glucan/chitin matrix
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
M – Mol. Weight markers
1. WT cell extract
2. WT secreted protein
3. Δcps-1 cell extract
4. Δcps-1 secreted protein
1
2
3
4
Δcps-1 releases cell wall protein into
the medium
Western blot analysis of ACW-1 cell wall protein
1
1.
2.
3.
4.
2
3
4
Δcps-1 secreted protein
Δcps-1 cell extract
WT secreted protein
WT cell extract
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 crosslink proteins into the cell wall.
• DFG5 and DCW1 are cell wall α-1,6mannanases/ 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-1
Eleanor Sokolow – cps-1
Ci 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
Δags-1 is affected in conidia
production
α-1,3-glucan is found in the conidial
cell wall.
The ags-1 5’regulatory DNA directs
expression in aerial hyphae and
developing conidia

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