title: A novel sorting motif in the glutamate transporter excitatory amino acid transporter 3 directs its targeting in Madin-Darby canine kidney cells and hippocampal neurons.
abstract: The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.
authors.affiliation: Howard Hughes Medical Institute, Oregon Health and Science University, Portland, Oregon 97239, USA.
authors.forename: Chialin
authors.initials: C
authors.lastname: Cheng
authors.forename: Greta
authors.initials: G
authors.lastname: Glover
authors.forename: Gary
authors.initials: G
authors.lastname: Banker
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D002478:Cells, Cultured; D003712:Dendrites / Q000737:chemistry; D004285:Dogs; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D006624:Hippocampus / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D051381:Rats; D027981:Symporters / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D021382:Protein Sorting Signals; C493398:Slc1a1 protein, rat; C493395:Slc1a2 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters