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A screen for spore wall permeability mutants identifies a secreted protease required for proper spore wall assembly.

Suda Y, Rodriguez RK, Coluccio AE, Neiman AM - PLoS ONE (2009)

Bottom Line: This dityrosine layer is important for stress resistance of the spore.Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer.These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

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Related in: MedlinePlus

osw mutant spores are sensitive to digestion by β-glucanases.AN120 (wild type) and the indicated osw mutant strains were sporulated and the survival of the spores assessed after exposure to Zymolyase. Percent survival for each strain was normalized to the average survival of the wild type strain. Data shown are the averages of at least four experiments. The vertical lines indicate one standard deviation.
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pone-0007184-g006: osw mutant spores are sensitive to digestion by β-glucanases.AN120 (wild type) and the indicated osw mutant strains were sporulated and the survival of the spores assessed after exposure to Zymolyase. Percent survival for each strain was normalized to the average survival of the wild type strain. Data shown are the averages of at least four experiments. The vertical lines indicate one standard deviation.

Mentions: The leakage assay reveals that spore wall blocks the passage of protein size molecules from the periplasmic space out of the spore wall. The wall also likely presents a similar barrier to the entry of protein-sized molecules. Vegetative cells are killed by prolonged exposure to β-glucanase enzymes. These enzymes digest the β-1,3 glucans within the cell wall leading to loss of wall rigidity and cell lysis. Spores are resistant to β-glucanase digestion, probably because the β-glucan layer of the spore wall is shielded by the outer wall layers. Therefore, to examine if the osw mutations also increased the permeability of the wall to exogenous proteins, the resistance of osw mutant spores to β-glucanase digestion was examined (Figure 6). Wild type, osw1Δ, osw3Δ, osw4Δ, and osw5Δ mutant strains were sporulated and spores were titered both before and after one hour of treatment with β-glucanases. All four mutants were more sensitive to digestion than the wild-type spores with survival ranging from 12% (osw1Δ) to 47% (osw5Δ) that of wild type. These results indicate that the increased permeability of these mutants allows movement of proteins both into and out of the spore wall.


A screen for spore wall permeability mutants identifies a secreted protease required for proper spore wall assembly.

Suda Y, Rodriguez RK, Coluccio AE, Neiman AM - PLoS ONE (2009)

osw mutant spores are sensitive to digestion by β-glucanases.AN120 (wild type) and the indicated osw mutant strains were sporulated and the survival of the spores assessed after exposure to Zymolyase. Percent survival for each strain was normalized to the average survival of the wild type strain. Data shown are the averages of at least four experiments. The vertical lines indicate one standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743993&req=5

pone-0007184-g006: osw mutant spores are sensitive to digestion by β-glucanases.AN120 (wild type) and the indicated osw mutant strains were sporulated and the survival of the spores assessed after exposure to Zymolyase. Percent survival for each strain was normalized to the average survival of the wild type strain. Data shown are the averages of at least four experiments. The vertical lines indicate one standard deviation.
Mentions: The leakage assay reveals that spore wall blocks the passage of protein size molecules from the periplasmic space out of the spore wall. The wall also likely presents a similar barrier to the entry of protein-sized molecules. Vegetative cells are killed by prolonged exposure to β-glucanase enzymes. These enzymes digest the β-1,3 glucans within the cell wall leading to loss of wall rigidity and cell lysis. Spores are resistant to β-glucanase digestion, probably because the β-glucan layer of the spore wall is shielded by the outer wall layers. Therefore, to examine if the osw mutations also increased the permeability of the wall to exogenous proteins, the resistance of osw mutant spores to β-glucanase digestion was examined (Figure 6). Wild type, osw1Δ, osw3Δ, osw4Δ, and osw5Δ mutant strains were sporulated and spores were titered both before and after one hour of treatment with β-glucanases. All four mutants were more sensitive to digestion than the wild-type spores with survival ranging from 12% (osw1Δ) to 47% (osw5Δ) that of wild type. These results indicate that the increased permeability of these mutants allows movement of proteins both into and out of the spore wall.

Bottom Line: This dityrosine layer is important for stress resistance of the spore.Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer.These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

Show MeSH
Related in: MedlinePlus