title: Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.
abstract: Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Rong
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan; WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hirata
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Motooka
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
authors.forename: Yi-Shi
authors.initials: YS
authors.lastname: Liu
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
authors.forename: Zeng-An
authors.initials: ZA
authors.lastname: He
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Gao
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan; WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Maeda
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan; WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Taroh
authors.initials: T
authors.lastname: Kinoshita
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
authors.forename: Morihisa
authors.initials: M
authors.lastname: Fujita
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005820:Genetic Testing; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism
chemical_list: D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins