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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

The dityrosine layer is essential for the Spr1-GFP diffusion barrier.AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were transferred to sporulation medium and, at the indicated time points after transfer, cells were collected and the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus was determined. More than 200 cells were analyzed at each time point. Values shown are the averages of three independent experiments. The vertical lines indicate the range of the values.
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pone-0007184-g002: The dityrosine layer is essential for the Spr1-GFP diffusion barrier.AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were transferred to sporulation medium and, at the indicated time points after transfer, cells were collected and the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus was determined. More than 200 cells were analyzed at each time point. Values shown are the averages of three independent experiments. The vertical lines indicate the range of the values.

Mentions: To determine if the outer layers of the spore wall serve as a barrier to retain Spr1-GFP in the wall, a plasmid carrying the same Spr1-GFP fusion was transformed into a chs3 mutant, which forms spores lacking both the chitosan and dityrosine layers, and a dit1 mutant, which forms spores lacking only the dityrosine layer [4], [8]. Spr1-GFP behavior was similar in both strains. Specifically, during prospore membrane formation and at early time points after prospore membrane closure, GFP fluorescence was limited to the prospore membrane compartment. However, the GFP subsequently diffused out of the spore wall and was retained in the ascal cytoplasm so that by twenty hours after transfer to sporulation medium, 100% of the cells displayed fluorescence throughout the ascus (Figure 1). A time course analysis of the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus suggested that the rate at which Spr1-GFP diffused out of the spore wall was comparable in both the chs3 and dit1 strains (Figure 2). Because the absence of the dityrosine layer alone leads to a similar rate of diffusion as the absence of both the chitosan and dityrosine layers, these data suggest that the dityrosine layer is the primary barrier to diffusion of Spr1-GFP from the 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)

The dityrosine layer is essential for the Spr1-GFP diffusion barrier.AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were transferred to sporulation medium and, at the indicated time points after transfer, cells were collected and the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus was determined. More than 200 cells were analyzed at each time point. Values shown are the averages of three independent experiments. The vertical lines indicate the range of the values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007184-g002: The dityrosine layer is essential for the Spr1-GFP diffusion barrier.AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were transferred to sporulation medium and, at the indicated time points after transfer, cells were collected and the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus was determined. More than 200 cells were analyzed at each time point. Values shown are the averages of three independent experiments. The vertical lines indicate the range of the values.
Mentions: To determine if the outer layers of the spore wall serve as a barrier to retain Spr1-GFP in the wall, a plasmid carrying the same Spr1-GFP fusion was transformed into a chs3 mutant, which forms spores lacking both the chitosan and dityrosine layers, and a dit1 mutant, which forms spores lacking only the dityrosine layer [4], [8]. Spr1-GFP behavior was similar in both strains. Specifically, during prospore membrane formation and at early time points after prospore membrane closure, GFP fluorescence was limited to the prospore membrane compartment. However, the GFP subsequently diffused out of the spore wall and was retained in the ascal cytoplasm so that by twenty hours after transfer to sporulation medium, 100% of the cells displayed fluorescence throughout the ascus (Figure 1). A time course analysis of the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus suggested that the rate at which Spr1-GFP diffused out of the spore wall was comparable in both the chs3 and dit1 strains (Figure 2). Because the absence of the dityrosine layer alone leads to a similar rate of diffusion as the absence of both the chitosan and dityrosine layers, these data suggest that the dityrosine layer is the primary barrier to diffusion of Spr1-GFP from the 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