Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

Abstract

Re-uptake of ␥-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.␥-Aminobutyric acid (GABA) 3 is the major inhibitory neurotransmitter in the mammalian brain. Its action is terminated by re-uptake into synaptic terminals. This is achieved by the GABA transporter 1 (GAT1), the predominant isoform in the central nervous system. GAT1 belongs to the Na ϩ /Cl Ϫ -dependent SLC6 gene family, which also includes transporters for serotonin (SERT), dopamine (DAT), and norepinephrine transporter. The turnover number of these transporters is low (i.e. in the range of 10 s Ϫ1 ). Accordingly, the number of active transporters at the cell surface is critical for the duration of synaptic transmission. This is evident from the gene dosage effect in mice engineered to be GAT1-deficient. The absence of one Gat1-allele homozygous suffices to produce phenotypic consequences: heterozygous mice Gat1 ϩ/Ϫ consume more ethanol, are more responsive to locomotor activation by ethanol, and are more prone to ethanol dependence than their wild type counterparts (1). Gat1 Ϫ/Ϫ mice, however, suffer from a complex motor disorder that includes abnormal gait, reduced locomotion, and tremor (2).It has been widely appreciated that the number of regulatory membrane proteins (that is receptor, transporters, and ion channels) on the surface is controlled by the rate of endocytosis, recycling, and proteosomal/lysosomal degradation (see e.g. Refs. 3 and 4). In contrast, the role of ER export has been explored to a lesser extent but there is evidence that the steadystate level of membrane proteins can also be determined by a rate-limiting step at the level of the ER (5). Originally, it was assumed that folding and quality control was the major dete...

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

“…These compounds effect recycling and internalization and thereby the density of SERT on the plasma membrane (21). The role of substrates in the trafficking of ␥-aminobutyric acid transporter (22,23), DAT (24), and the excitatory amino acid transporter 1 is to regulate the expression of the transporter on the cell mem-brane, i.e. glutamate increases the surface expression of excitatory amino acid transporter 1 (25).…”

Di-acidic Motifs in the Membrane-distal C Termini Modulate the Transport of Angiotensin II Receptors from the Endoplasmic Reticulum to the Cell Surface

“…It has been demonstrated that di-acidic motifs are involved in rapid concentration of cargo into the COPIIcoated vesicles and are able to directly bind to the COPII component Sec24 (1,2,6,13,55,56). We have demonstrated here that the function of the ExD motif in modulating AT2R transport to the cell surface is likely mediated through enhancing the recruitment of the cargo onto ER export sites or the COPII transport vesicles.…”

“…This interaction results in the concentration of cargoes in the ER exit sites and facilitates cargo recruitment onto the COPII vesicles (13).…”

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…This is likely due to a slow ER exit for this mutant. Indeed, a role for K590 and L591 in sec24D binding and ER export has recently been reported for DAT, SERT and GAT (Farhan et al, 2007). It is interesting that the highly conserved K590 appears to play an important trafficking role, both at the level of ER exit and for regulated endocytosis.…”