Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

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 biosy...

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Bibliographic Details
Published in:PloS one 2015-09, Vol.10 (9), p.e0138553
Main Authors: Rong, Yao, Nakamura, Shota, Hirata, Tetsuya, Motooka, Daisuke, Liu, Yi-Shi, He, Zeng-An, Gao, Xiao-Dong, Maeda, Yusuke, Kinoshita, Taroh, Fujita, Morihisa
Format: Article
Language:English
Subjects:
Biosynthesis
Biotechnology
Carbohydrates
CD59 antigen
Cloning
CRISPR
Education
Endoplasmic reticulum
Endoplasmic Reticulum - metabolism
Enrichment
Gene expression
Gene sequencing
Genes
Genetic screening
Genetic Testing
Genomes
Genomics
Glycosylphosphatidylinositol
Glycosylphosphatidylinositols
Glycosylphosphatidylinositols - biosynthesis
Golgi apparatus
Golgi Apparatus - metabolism
Humans
Immunology
Insertion
Laboratories
Mammals
Medical screening
Membrane Proteins - metabolism
Methods
Mutation
Phenotypes
Pore formation
Protein binding
Proteins
R&D
Research & development
Rodents
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0138553