title: Concentrative export from the endoplasmic reticulum of the gamma-aminobutyric acid transporter 1 requires binding to SEC24D.
abstract: Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.
authors.affiliation: Institute of Pharmacology and Institute of Vascular Biology and Thrombosis Research, Center of Biomolecular Medicine and Pharmacology, Waehringer Strasse 13a, A-1090 Vienna, Austria.
authors.forename: Hesso
authors.initials: H
authors.lastname: Farhan
authors.forename: Veronika
authors.initials: V
authors.lastname: Reiterer
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Korkhov
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Schmid
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000502:physiology*
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; C530641:SEC24D protein, human; C493413:SLC6A1 protein, human; D033921:Vesicular Transport Proteins