The Effect Of N-terminal Domain Of Rat Glutamine Transporters SNAT2on Locating To Cell Membranes

Sodium-coupled neutral amino acid transporters (SNATs) in the central neural systemplayed an impotrant role in transporting small, neutral amino acids, such as glutamineand alanine, across cellular membranes. In order to detect expression and localizationof SNAT2on cell membranes conveniently，an EGFP protein sequence was subclonedinto SNAT2’s C-terminus in the eukaryotic expression vector pBK-CMV-(A[l098-1300])-SNAT2-myc. After pBK-CMV-(A[l098-1300])-SNAT2-EGFP was transientlytransfected into HEK293T cells, expression and localization on cell membranes ofSNAT2-EGFP was detected by laser scanning confocal microscope and Western blotusing anti-GFP antibody. In order to study the effect of N-terminal domain of SNAT2on locating to cell membranes, the M1G-SNAT2-EGFP and SNAT2Dei N-te-rEGFPwere obtained by removing5amino acid residues of N-terminal of SNAT2bysite-directed mutagenesis and by removing75amino acid residues of entireN-terminal of SNAT2by restructuring vector, respectively. Expression ofM1G-SNAT2-EGFP and SNAT2Dei N-ter-EGFP were detected by laser scanningconfocal microscope and Western blot using anti-GFP antibody. The mRNAs level ofM1G-SNAT2-EGFP and SNAT2Dei N-terEGFP were detected by RT-PCR. The resultsshowed that SNAT2-EGFP was successfully expressed and localized on cellmembranes. In transiently mutant transporters-transfected human embryonic kidney293T cells, M1G-SNAT2-EGFP expressed and located to cell membranes normally asSNAT-EGFP. However, the expression of SNAT2Dei N-te-rEGFP was dramaticallyreduced compared to SNAT-EGFP or M1G-SNAT2-EGFP. It also couldn’t locate tocell membranes. There was no difference in the expression level of mRNA of SNAT2Dei N-te-rEGFP,SNAT-EGFP and M1G-SNAT2-EGFP. We concluded that N-terminal of SNAT2plays an important role in regulation of membrane expression and location atranslational level.