Characterization Of TMEM208 Involved In ER Entry Of GPI-anchored Proteins In Mammalian Cells

Glycosylphosphatidylinositol-anchored modification is a conserved post-translational modification in eukaryotic cells.In mammalian cells,more than 100 proteins expressed on cell surface are modified by GPI anchors.In the biosynthesis of GPI-anchored proteins,nascent proteins are synthesised in the ribosome,transferred into the endoplasmic reticulum(ER),and attched to the GPI anchor with GPI-anchor transamidase complex.Nascent GPI-anchored proteins(GPI-APs)experience lipid remodeling in the ER and after transporting to the Golgi,then expressing on the cell surface.However,the specific mechanism of nascent proteins transferred into the ER from ribisome remains unclear.In mammalian cells,nascent proteins enter the ER through SRP,GET,or SEC62/63 pathway.However,GPI-anchored proteins do not rely on these pathways completing the ER translocation.Recently,SND(SRP-independent targeting)pathway consisting of Snd1,Snd2,and Snd3,is identified as a new ER translocation pathway in yeast.At the same time,TMEM208 is identified as the homologue of Snd2 in mammalian cells.Previous paper reported that TMEM208 mediates the nascent proteins with C-terminal transmembrane domain into the ER lumen.In this paper,TMEM208 deficiency led to 50% decreased expression of GPI-APs(CD59,DAF and CD109),but did not change the levels of non-GPI type-I membrane proteins(BSG and CD49e).Decreased expression of GPI-AP in TMEM208-KO cells was not due to defects in either m RNA levels or GPI processing.SRPRA,SRPRB,and TMEM208 were involved in the ER targeting of CD59 in mammalian cells.Coimmunoprecipitation and immunoblotting showed that TMEM208 associating with SEC61 translocon complex mediated the GPI-APs translocon process from ribosome to the ER.The deletion or mutation of mature peptide did not change GPI-APs expression in TMEM208-KO cells.When carrying the identical N-terminal signal peptide,different GPI-anchored proteins show distict expression in HEK293 and TMEM208-KO cells,which indicated that the C-terminal GPI-anchored signal peptide(GPIAS)of GPI-anchored proteins are required for TMEM208-mediated ER targeting.When carrying strong hydrophobic GPIAS,GPI-APs expression was not affected in TMEM208-KO cells;when carrying week hydrophobic GPIAS,GPI-APs expression decreased in TMEM208-KO cells.Therefore,we concluded that TMEM208 recognized the N-terminal and C-terminal signal peptides of GPI-APs,and the hydrophobicity of GPIAS was capable of determination of TMEM208 dependency when N-terminal SS has TMEM208 dependency.Taken together,our results revealed the function of TMEM208 in mammalian cells,identify the mechanisms of TMEM208 in the ER translocation of GPI-anchored proteins,contribute to diseases treatment and therapy development.