Abstract
The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.
Highlights
Glycosylphosphatidylinositol (GPI) is a complex glycolipid that acts as a membrane anchor for many cell surface proteins [1,2,3] and is a highly conserved post-translational modification from yeast to mammals
The calnexin/ calreticulin cycle is essential for N-glycosylated protein folding [23], and GPI-inositol deacylation is required for the efficient sorting of glycosylphosphatidylinositol-anchored protein (GPI-AP) into transport vesicles and their subsequent transport from endoplasmic reticulum (ER) to the Golgi apparatus [8, 43]
Folded and inositol-deacylated GPI-APs are further processed by PGAP5 and recognized by p24 cargo receptors to become incorporated into transport vesicles
Summary
Glycosylphosphatidylinositol (GPI) is a complex glycolipid that acts as a membrane anchor for many cell surface proteins [1,2,3] and is a highly conserved post-translational modification from yeast to mammals. In calnexin and calreticulin double-knockout (CANX&CALR-DKO) cells, protein folding and GPI-inositol deacylation efficiencies were significantly decreased such that misfolded and inositol-acylated GPI-APs were exposed on the plasma membrane. To determine whether calnexin/calreticulin affects the inositol deacylation of non-N-glycosylated GPI-APs, we analyzed LY6D and GFP-GPI, which have no N-glycan, by PI-PLC treatment in WT and CANX&CALRDKO cells.
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