ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.