Functional Study Of Glycosylation Modification Of Amino Acid Transporter PAT1

PAT1 is a conserved amino acid transporter in vertebrates.It is mainly located on the cell surface and lysosome,and plays a role in metabolism and the mTORC1 signaling pathway.Using Xenopusoocyte as a model,Dorn et al.found that the human PAT1 protein was mainly glycosylated on three asparagine residues in its extracellular regions(N174,183,470).They further demonstrated that glycosylation was not important for the stability of PAT 1.Instead,the unglycosylated form of PAT1(PAT13NQ)faield to be expressed on the cell surface.Based on these findings,we suspected PAT13NQ might be relocatedto the lysosome,and thereby inhibited mTORC1.To test this hypothesis,we generated HEK293 cell lines stablely expressing EGFP-PAT13NQ.Using the cell line as a model,we obtained the following results:1.Overexpression of EGFP-PATl3NQ did not inhbit mTORCl;2.The EGFP-PAT13NQ protein was unstable,it was mainly degraded via the ERAD pathway;3.Compared with the wild-type EGFP-PAT1,the lysosomal expression of EGFP-PAT13NQ was decreased and the cell surface expression of EGFP-PAT13NQ was increased.This altered subcellular localization provides an explanation of why overexpression of EGFP-PAT13NQ did not inhibit mTORC1.Taken together,our data demonstrate that glycosylation modification plays two roles on PAT1 in HEK293 cells.First,it protects PAT1 protein from degradation;second,it affects the distribution of PAT 1 on the cell surface and lysosome,and thereby regulates the mTORC1 activity.