Endoplasmic Reticulum-Associated Degradation Pathway Analysis Of GPI Anchored Protein Precursor

GPI(glycosylphosphatidylinositol)anchoring is one of the post-translational modification ways in eukaryotes.more than 150 glycosylphosphatidylinositol-anchored proteins(GPI-APs)are expressed in the mammalian cells and involved in various physiological processes such as cell communication,immune recognition and signal transduction.In the case of proteins persistent-misfolding caused by the extreme environment,misfolded/unfolded proteins can be degraded by endoplasmic reticulum-associated degradation(ERAD).ERAD includes three ways: ERAD-C,ERAD-L and ERAD-M,which are responsible for the degradation of misfolded/unfolded proteins in the cytoplasm,in the ER lumen and on the ER membrane,respectively.GPI is transferred to proteins in the endoplasmic reticulum(ER),which catalyzed by GPI transamidase.When GPI-anchoring is impaired,precursor proteins are thought to be degraded through ER-associated degradation(ERAD).However,the mechanism of their degradation in ERAD remains unclear.Here,we found that the degradation of GPI precursor proteins depends on two ERAD pathways: ERAD-L and ERAD-M pathways.Two E3 ubiqutin ligases responsible for the pathways,HRD1 for ERAD-L and GP78 for ERAD-M were selectively utilized for GPI protein precursor degradation.The hydrophobicity of GPI C-terminal attachment signal on precursor proteins was correlated to the selection of the pathways.Neither the lectin OS9 nor N-glycans on proteins affected in degradation of HRD1-dependent GPI precursor proteins.Our results explain the mechanism of ERAD degradation pathways for unprocessed GPI precursor proteins in mammalian cells,which provides a good reference value for future disease treatment and drug development.Many biomolecules can be detected through the strong reaction of biotin and streptavidin.This widely used method is called biotin labeling.This study proved that proximity labeling method is applicable to study the ERAD degradation pathway of GPI-anchored protein precursors.Furthermore,biotin ligase Turbo ID-KDEL can be stably expressed in knockout cell lines with accurate localization.In addition,this study also made some progress in the exploration of the optimal conditions for Turbo ID-KDEL,such as optimal incubation time for biotin.The results of protein identification by mass spectrometry were satisfactory,which laid a good foundation for the subsequent analysis,and established a set of productive methods for future application.