title: N-linked glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane.
abstract: The human amino acid transporter SLC1A5 (ASCT2) contains two N-glycosylation sites (N163 and N212) located in the large extracellular loop. In the homology structural model of ASCT2 these Asn residues are extracellularly exposed. Mutants of the two Asn exhibited altered electrophoretic mobility. N163Q and N212Q displayed multiple bands with apparent molecular masses from 80kDa to 50kDa. N163/212Q displayed a single band of 50kDa corresponding to the unglycosylated protein. The presence in membrane of WT and mutants was evaluated by protein biotinylation assay followed by immunoblotting. The double mutation significantly impaired the presence of the protein in membrane, without impairment in protein synthesis. [(3)H]glutamine transport was measured in cells transiently transfected with the WT or mutants. N163/212Q exhibited a strongly reduced transport activity correlating with reduced surface expression. The same proteins extracted from cells and reconstituted in liposomes showed comparable transport activities demonstrating that the intrinsic transport function of the mutants was not affected. The rate of endocytosis of ASCT2 was assayed by a reversible biotinylation strategy. N212Q and N163/212Q showed strongly increased rates of endocytosis respect to WT. ASCT2 stability was determined using cycloheximide. N163Q or N163/212Q showed a slightly or significantly lower stability with respect to WT. To assess trafficking to the membrane, a brefeldin-based assay, which caused retention of proteins in ER, was performed. One hour after brefeldin removal WT protein was localized to the plasma membrane while the double mutant was localized in the cytosol. The results demonstrate that N-glycosylation is critical for trafficking.
authors: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy; Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto M5B 2K3, Canada.
authors: Lara
authors: L
authors: Console
authors: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Mariafrancesca
authors: M
authors: Scalise
authors: Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto M5B 2K3, Canada.
authors: Zlatina
authors: Z
authors: Tarmakova
authors: Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto M5B 2K3, Canada.
authors: Imogen R
authors: IR
authors: Coe
authors: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Cesare
authors: C
authors: Indiveri
mesh_terms: D026962:Amino Acid Transport System ASC / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D019295:Computational Biology; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008958:Models, Molecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D051381:Rats; D040681:Structural Homology, Protein; D013997:Time Factors
chemical_list: D026962:Amino Acid Transport System ASC; D015778:Minor Histocompatibility Antigens; C420506:SLC1A5 protein, human; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Glutamine; Liposomes; Plasma membrane; Protein stability; Transport