title: Two discontinuous segments in the carboxyl terminus are required for membrane targeting of the rat gamma-aminobutyric acid transporter-1 (GAT1).
abstract: Like all members of the Na(+)/Cl(-)-dependent neurotransmitter transporter family, the rat gamma-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Delta37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Delta27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Delta27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Delta3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Delta37 and GAT1-Delta32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif ((569)VMI(571)) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.
authors.affiliation: Institute of Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
authors.forename: Hesso
authors.initials: H
authors.lastname: Farhan
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Korkhov
authors.forename: Verena
authors.initials: V
authors.lastname: Paulitschke
authors.forename: Mario M
authors.initials: MM
authors.lastname: Dorostkar
authors.forename: Petra
authors.initials: P
authors.lastname: Scholze
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019921:Biotinylation; D002352:Carrier Proteins / Q000737:chemistry*; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D050485:GABA Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors; D014162:Transfection
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins