Concentrative export from the endoplasmic reticulum of the γ-aminobutyric acid transporter 1 requires binding to SEC24D

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 GATI ((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 GATI: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (DD734)-D-733 by (VN734)-V-733), and mutation of (RL567)-R-566 to (566)AS(567) (GATI-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT 1 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 GATI. Our observations show that concentrative ER-export is contingent on a direct interaction of GATI 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.