Study On Regulatory Mechanism For The Lipid Scramblase TMEM41B In The Processing And Transport Of GPI-anchored Proteins

Glycosylphosphatidylinositol(GPI)-anchoring is a conserved post-translational modification of proteins in eukaryotic cells.The deacylation of GPI anchor is performed in the endoplasmic reticulum(ER),which is catalyzed by the GPI-inositol deacylase post-GPI attachment protein 1(PGAP1)after GPI attachment to ancient proteins.The correct structure of GPI anchor is crucial for efficient traficking to the Golgi through transport vesicles.The ER scramblase TMEM41 B transport lipid nonspecifically to regulate membrane stability and asymmetry,which contributes to various dynamic remodeling of ER membranes.Here,the roles of TMEM41 B in the deacylation of GPI anchor were addressed.On the one hand,TMEM41 B deficiency delayed the exit of GPI-APs from ER,on the other hand,its deficiency stablized PGAP1,contributing accumulation of PGAP1 in the ER.The increased ER retention time of GPI-APs and an increased level of PGAP1 would result in more efficient GPI-inositol deacylation.The main results of the present study are summarized as follows:(1)CRISPR-Cas9 based genetic screening identified TMEM41 B as a factor that affected GPIinositol deacylation.Previous results showed most of GPI-APs were expressed on cell surface as inositol-acylated form in SELT-KO cell.In this research,a genome-wide CRISPR-Cas9 based genetic screening was performed and it was found that TMEM41 B deficiency rescued the phenotype of inositol-deacylation of GPI-APs in SELT-KO cell.Futher verification showed TMEM41 B worked as a general factor,deficiency of which increased the efficiency of GPI-inositol deacylation both in HEK293 and SELT-KO cells.(2)TMEM41B involved in the transport of GPI-APs and transmembrane proteins from the ER to the Golgi.By way of immunoblotting and confocal immunoflurorescent staining technique,the expression of GPI-APs was increased and the ER form was accumulated in TMEM41B-KO cell.The temperature-sensitive reporter proteins with GPI anchor or transmembrane domain were used to monitor protein transport,and both of them became slower after knocking out TMEM41 B.PGAP1catalyzes the cleavage of the acyl-chain linked to inositol ring.In PGAP1-KO cell,the inositol acylated GPI-APs can’t exit from the ER efficiently.TMEM41 B deficiency decreased the transport futher in PGAP1-KO cell,which indicated the TMEM41 B and PGAP1 affected the transport of GPIAPs synergistically.Taken those results together,TMEM41 B deficiency impaired the transport of GPI-APs and transmembrane proteins from the ER to the Golgi.(3)TMEM41B deficiency stabilized human PGAP1.The protein of PGAP1 accumulated in the TMEM41B-KO cell without transcription changes.To detecte the protein degradation machnism,the proteasome and autophagy mediated protein degradation pathways were inhibited,as well as knocking out E3 ubiquition lipase,the results indicated the degradation of PGAP1 was dependent on the ER related protein degradation pathway mediated by proteasome and GP78.The protein chase assay showed PGAP1 degradation impaired in TMEM41B-KO cell.Taken together,the stabilization of PGAP1 in TMEM41B-KO cell was increased,which contributed to the efficiency of GPI-inositol deacylation and rescued the protein level of deacylated GPI-APs on plasma membrane in SELT-KO cell.