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.
Highlights
Dependent SLC6 gene family, which includes transporters for serotonin (SERT), dopamine (DAT), and norepinephrine transporter
The transporter of interest must be trapped in the endoplasmic reticulum (ER): if the leucine repeat in transmembrane segment 2 of GABA transporter 1 (GAT1) is disrupted by substitution with alanines, the resulting GAT1-L2A is defective in oligomerization and retained in the ER [12]
We ruled out that the lack of Sec24D binding by GST-RL/AS was due to a mutation-induced misfolding of the COOH terminus by carrying out pulldown experiments with YFP-Pals1, a protein that interacts with the last three amino acids of GAT1 [19]
Summary
Reagents, and Mutagenesis—The plasmid encoding YFP-Sec24D was a gift from R. In the case of budding assays, cells were transiently transfected using Lipofectamine Plus (Invitrogen). For fluorescence resonance energy transfer (FRET) microscopy, HEK293 cells were transfected with mutated versions of CFP-tagged GAT1 and with YFP-tagged Sec24D as indicated in the figure legends. HEK293 cells were transfected with plasmids encoding YFP-tagged Sec24D or YFP-tagged Pals-1. In Vitro Budding Assay—HEK293 cells in 10-cm culture dishes were transfected with the plasmids specified in the pertinent figure legends using Lipofectamine Plus. The membrane was incubated with horseradish peroxidase-tagged anti-rabbit antibody (diluted 1:10,000 in TBST with 5% milk) for 1 h at room temperature. The cytosol employed in these complementation experiments was prepared from control cells and from appropriately transfected cells (4 ϫ 107) by sonication in 0.4 ml of buffer (20 mM Tris-HCl, pH 7.2, 130 mM KCl). The particulate material was removed by centrifugation (70 min at 40,000 ϫ g)
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