Expression And Identification Of Glutamine Transporter EGFP - SNAT3 Fusion Protein And Analysis Of SNAT2 Topological Structure

The sodium-coupled neutral amino acid transporters(SNATs) belong to the SLC38 family. SLC38 family is divided into System A and System N in terms of functional properties and patterns of regulation. SNAT2 belongs to System A,SNAT3 belongs to System N. Both SNAT2 and SNAT3 consist of 504 amino acids,and the theoretical molecular weight is about 56 k Da. They mediate Na+/amino acid cotransport and the transport process is p H sensitive. SNAT2 is the second member of System A and widely expressed in mammalian tissues. It has been reported that the alterations in SNAT2 is responsible for several neurodegenerative disorders.SNAT3 is encoded by SLC38A3, its expression in the brain is largely confined to the plasma membrane in astrocytes. SNAT3 involves H+ exchange as well as Na+cotransport. SNAT2 and SNAT3 play important roles in glutamate-glutamine cycle,gluconeogenesis and ammonia detoxification in the brain and the liver. Given their important physiological functions, it will be of great significance to explore their structure and functions in depth.In order to detect expression and localization of SNAT3 on the membrane conveniently, in this study, an enhanced green fluorescent protein(EGFP)sequence was constructed to the N terminus of SNAT3 by PCR amplification,enzyme digestion and ligation. After the recombinant eukaryotic expression plasmid of p BK-CMV(Δ[1098-1300])-EGFP-SNAT3 was transiently transfected into HEK293 T cells mediated by liposomal for 36 hours, expression and localization of EGFP-SNAT3 fusion protein were detected by laser scanning confocal microscope and Western blot. The results showed that EGFP-SNAT3 expressed and localized correctly on the membrane. The recombinant plasmid of p BK-CMV(Δ[1098-1300])-EGFP-SNAT3 constructed successfully may contribute to the further study of structure and function of SNAT3 in the future.Hydrophobic analysis predicts that SNAT2 contains 11 transmembrane segments(TMSs) with an intracellular N terminus and an extracellular C terminus.In this study, we determined the topology structure of SNAT2(the memebrane localization of the amino and carboxyl termini, the number and distribution of TMS) by inserting hemagglutinin(HA) tags in the predicted hydrophilic regions of SNAT2. Seventeen HA-tagged SNAT2 mutants were transiently transfected intoHEK293 T cells mediated by liposomal, respectively. Position of the inserted tags with respect to the plasma membrane( intracellular or extracellular) was then determined by indirect immunofluorescence in intact and permeabilized cells using an HA tag-specific primary antibody and an Alexa Fluor 488 labeled HA fluorescent secondary antibody. Meanwhile, we determined the memebrane localization of the amino and carboxyl termini of SNAT2 by immunofluorescence.The results provide the first direct experimental evidence in support of a 10-TMS model for SNAT2 with the amino and carboxyl termini located extracellularly.