Construction Of Rat Neutral Amino Acid Transporter HA-SNAT3 Fusion Protein Identification Of Disulfide Bonds In SNAT1 And SNAT2

Sodium-coupled Neutral Amino Acid Transporter（SNAT） belongs to the Solute Carrier 38 family（SLC38）, which contains 11 members, namely SNAT1-11. They are ubiquitously expressed in mammalian tissues and have critical physiological functions.SNAT3 is a classical member of System N, which transports Gln and His predominantly. SNAT3 has the ability to counter transport both Na⁺ and H⁺, and reveals tolerance to replacement of Na⁺ by Li⁺. Abundant expression of SNAT3 protein is detected in astrocytes throughout the retina and brain. It has been suggested to be responsible for glutamine transport in glutamate-glutamine cycle in the brain and liver. SNAT3 contains 504 amino acid residues with a predicted molecular mass of 55 k D. In order to easily detect the expression and localization of SNAT3 on the membrane, we constructed a eukaryotic expression plasmid with an HA tag at the N-terminus of rat SNAT3 in the mammalian expression vector p BK-CMVΔ（1098-1300） by PCR and double restriction endonuclease digestion techniques. After transiently transfected the plasmid into human embryonic kidney cells（HEK293T）, expression of HA-SNAT3 fusion protein was detected by Western bolt. The result suggested that the HA-SNAT3 fusion protein could normally express on the membrane. The plasmid p BK-CMVΔ（1098-1300）-HA-SNAT3 will provide a useful tool for the investigation of structure and function of SNAT3 in the future.SNAT1 and SNAT2 are members of System A, which are responsible for transporting small, aliphatic amino acids. They transport substrates coupled to the uptake of Na with a stoichiometry of 1:1. Both SNAT1 and SNAT2 are involved in the significant glutamate/glutamine cycle in neurons. The dysfunction of SNAT1 and SNAT2 may cause some neurodegenerative diseases.There are 10 cysteine residues in SNAT1 and 7 in SNAT2. In this study, we performed thiol-specific chemical modification by sequential treatment of NEM, DTT, and m PEG₅000-Mal to wildtype, 10 single C to A mutants and the C245 A,C262A double mutant of SNAT1. The results of Western blot suggested that the apparent molecular weight of single mutants C245 A and C262 A were shifted to about 120 k D, as well as SNAT1 wildtype. On the contrary, the protein molecular weight of 8 other single mutants and C245 A,C262A double mutant remained to be 55 k D. The results suggested that Cys245 and Cys262 formed a disulfide bond in SNAT1. The same way of chemical modification was used to identify the disulfide bridge in SNAT2. The results indicated that the apparent molecular weight of SNAT2 wildtype and C245 A and C262 A single mutants was shifted to about 110 k D, while that of 5 other single mutants and C245 A,C279A double mutant was still 55-70 k D. The disulfide bond in SNAT2 was thus identified between Cys245 and Cys279. Besides, the results of Immunofluorescence staining showed that both the cysteine residues and the disulfide bridge in SNAT1 and SNAT2 had no effect on their trafficking to the cell surface.The successful identification of disulfide bonds in SNAT1 and SNAT2 will be helpful to the study of their structure and function. It also provides an excellent method to identify the disulfide bond in other transmembrane proteins.