a screen for spore wall permeability mutants identifies a secreted protease required for proper spore wall assembly屏幕的孢子壁渗透率突变体识别分泌蛋白酶需要适当的孢子壁组装.pdfVIP

A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly Yasuyuki Suda¤a, Rachael K. Rodriguez, Alison E. Coluccio¤b, Aaron M. Neiman* 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. Citation: Suda Y, Rodriguez RK, Coluccio AE, Neiman AM (2009) A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly. PLoS ONE 4(9): e7184. doi:10.1371/journal.pone.0007184 Editor: Gustavo Goldman, Universidade de Sao Paulo, Brazil Received June 3, 2009; Accepted September 4, 2009; Published Se