N-Glycosylation and GPI anchoring of proteins occur in the endoplasmic reticulum (ER). Liu et al. revealed N-glycans participate in quality control and temporal ER retention of GPI-anchored proteins (GPI-APs), ensuring their correct folding and GPI processing before exiting from the ER. Chronic ER stress induced exposure of unprocessed GPI-APs on the cell surface.
Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.
Glycosylphosphatidylinositol (GPI) is a glycolipid added to the C-terminus of a large variety of proteins in eukaryotes, thereby anchoring these proteins to the cell surface. More than 150 different human proteins are modified with GPI, and GPI-anchored proteins (GPI-APs) play critical roles in embryogenesis, neurogenesis, immunity, and fertilization. GPI-APs are biosynthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the Golgi apparatus. During transport, GPI-APs undergo structural remodeling that is important for the efficient folding and sorting of GPI-APs. Asparagine-linked glycan-dependent folding and deacylation by PGAP1 work together to ensure that correctly folded GPI-APs are transported from the ER to the Golgi. Remodeling of the GPI lipid moiety is critical for the association of GPI-APs with lipid rafts. On the cell surface, certain GPI-APs are cleaved by GPI cleavage enzymes and released from the membrane, a key event in processes such as spermatogenesis and neurogenesis. In this review, we discuss the enzymes involved in GPI-AP biosynthesis and the fate of GPI-APs in mammalian cells, with a focus on the assembly, folding, degradation, and cleavage of GPI-APs.
Over 100 glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI‐APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)‐dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI‐APs to the ER. Both the N‐terminal signal sequence and C‐terminal GPI attachment signal of GPI‐APs contribute to ER targeting via the hSND2‐dependent pathway. Particularly, the hydrophobicity of the C‐terminal GPI attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI‐APs in mammalian cells.
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