Efficient transport of GPI-APs requires structural remodeling of GPI anchors by PGAP1 and PGAP5. (A) Flow cytometric analysis of transport of GPI-anchored reporter protein. Parental FF8 (WT), pgap1 mutant FPRC2, and pgap5 mutant C19 cells expressing VFG-GPI were stained with an anti-Flag antibody at the indicated times after a temperature shift from 40°C to 32°C. The percentages of cells in the pentagonal region are indicated. (B and C) The cell surface expression of VFG-GPI was analyzed by flow cytometry. FF8 (WT), FPRC2 (pgap1), C19 (pgap5), and FPRC2 cells stably expressing PGAP1 (+ PGAP1) and C19 cells stably expressing HA-tagged PGAP5 (+HA-PGAP5) were stained with anti-Flag antibody at the indicated times after the commencement of transport. The geometric mean fluorescence values of surface VFG-GPI in all cells shown in histograms (B) were quantified at each time point (C). The geometric mean of WT cells at 60 min was plotted as 100% relative transport. Values represent mean ± SD (error bars; n = 3). (D) Western blotting analysis of DAF in the steady state. Cell lysates of FF8 (WT), FPRC2 (pgap1), C19 (pgap5), and FPRC2 cells stably transfected with PGAP1, and C19 cells stably transfected with HA-PGAP5 were analyzed with an anti-DAF antibody under nonreducing conditions to evaluate the steady-state levels of the ER and mature forms. TfR, transferrin receptor for loading assessment.