Mol Cell Biochem. cells were analyzed by deep sequencing. Enrichment rates were calculated for the respective genes by comparing the numbers of mapped independent-insertion sites of the respective gene between sort 2 and control cells. The genes and (gene, which is required for GPI fatty acid remodeling in the Golgi (Tashima mutant cells, transport of GPI-APs is almost normal, but the surface expression of GPI-APs is greatly decreased because lyso form GPI-APs, the intermediate forms during the fatty acid Borussertib remodeling, which harbor only a single acyl chain, are not reacylated due to the PGAP2 defect and are released easily from the membrane into medium soon after arrival at the cell surface. Open in a separate window FIGURE 1: Haploid genetic screening of the factors required for the anterograde transport of GPI-APs. (A) Transport assay of VFG-GPI in HAP1FF9 wild-type cells (left) and the enriched cell population after sorting twice for delayed transport (right). (B) The significance of the enrichment of gene-trap insertions in an enriched population with delayed transport compared with the nonselected population was calculated TLN2 and plotted as a bubble plot. The horizontal line shows the chromosomal position of the genes, and the vertical line shows the significance of enrichment of each gene (value). The size of the bubble shows the number of inactivated insertion sites. Genes significantly enriched in sort 2 (< 0.01) are colored. Red bubbles, genes encoding the GARP complex subunit; green bubbles, genes involved in GPI-AP remodeling. The bubble of VPS53 indicated by an arrow was close to the significance limit. Knockout of GARP complex subunits severely impairs anterograde transport of proteins Three genes(also known as or < 0.01), and the fourth subunitVPS53was positioned close to the significance limit (Figure 1B and Supplemental Table S1). In addition, the Borussertib gene, encoding a subunit of conserved oligomeric Golgi (COG) complex, another multisubunit tethering factor, was highly enriched. To investigate whether the GARP complex is involved in the anterograde transport of GPI-APs, we generated GARP-KO cells using the clustered, regularly interspaced, short palindromic repeat (CRISPR)CCas9 system (Cong = 3). Post-Golgi anterograde transport is defective in V54KO cells The step in anterograde transport that is affected by a defect in the GARP complex was identified using confocal microscopy in cells stably transfected with an empty vector or VPS54 (V54KO+Vec or V54KO+VPS54), together with red fluorescent protein (RFP)CGPP34 as a TGN marker. VFG-GPI transport was chased for the indicated time, after which the cells were fixed to terminate the transport. Ratios of VFG-GPI fluorescence intensity in the TGN to total cellular VFG-GPI fluorescence intensity were determined at each time point (Figure 3B). In V54KO+VPS54 cells, the amount of VFG-GPI that reached the TGN gradually increased for 20 min, remained constant for the next 60 min, and then finally decreased at 90 min (Figure 3, A and B), suggesting that after VFG-GPI reached the TGN, it was transported to the cell surface. In V54KO+Vec cells, Borussertib the relative intensity in TGN was slightly increased at 20 min but further increased for the next 60 min (Figure 3, A and B), suggesting that after its arrival at the TGN, VFG-GPI accumulated in the Borussertib TGN for at least 60 min. This result suggested that GARP complex is required for post-Golgi transport. Although ER-to-Golgi transport appeared to be slightly impaired in V54KO+Vec cells (0.15 0.012 vs. 0.095 0.0089 at 20 min; mean SEM), this might have been because of the.