Two Discontinuous Segments in the Carboxyl Terminus Are Required for Membrane Targeting of the Rat γ-Aminobutyric Acid Transporter-1 (GAT1)

Abstract

Like all members of the Na؉ /Cl ؊ -dependent neurotransmitter transporter family, the rat ␥-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-⌬37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-⌬27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-⌬27 reached 50% of the wild type level. Additional extensions up to the position ؊3 (GAT1-⌬3) 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-⌬37 and GAT1-⌬32; 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.Neurotransmission at synaptic junctions in the central nervous system is terminated by reuptake of the neurotransmitter into the synaptic ends (1). This is achieved by neurotransmitter transporters. GABA 1 is the major inhibitory neurotransmitter in the brain. There are three GABA transporters, referred to as GAT1, GAT2, and GAT3, which all belong to the Na ϩ /Cl Ϫ -dependent neurotransmitter transporter family. Members of this family share several features including a characteristic topology, i.e. intracellular amino and the carboxyl termini and a hydrophobic core composed of 12 transmembrane-spanning segments that are presumed to be predominantly ␣-helical. In the brain, GAT1 is the most widely distributed isoform. The transporter is of obvious therapeutic relevance: increases in synaptic GABA reduces excitability and prevents excessive neuronal firing. This action provides a rationale for the use of tiagabine in the treatment of epilepsy (2). In neurons, transporters must reach the presynaptic specialization. Thus, their biosynthetic pathway must also comprise mechanisms that afford the sorting and targeting to the axonal compartment and/or specific retention at perisynaptic sites (3). In addition, they undergo quality control in the endoplasmic reticulum (4) and posttranslational modification in the Golgi stacks. We recently demonstrated that neurotransmitter transporters such as GAT1 and the serotonin transporter form constitutive oligomers (5). If oligomerization of GAT1 is disrupted, the transporter is no longer expressed at the cell surface but is retained ...

TheCaenorhabditis elegans snf-11Gene Encodes a Sodium-dependent GABA Transporter Required for Clearance of Synaptic GABA

“…The C-terminal cytoplasmic tail of rat GAT1 contains two important localization motifs: a hydrophobic VMI tripeptide (VMV in the human protein), and the C-terminal AYI, which is a type II PDZ domain interaction motif (Farhan et al, 2004). We note that SNF-11 contains neither of these motifs.…”

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

“…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. Fluorescence resonance energy transfer was measured by evaluating the extent to which fluorescence emitted by CFP increased after bleaching of YFP (DRAP, donor recovery after acceptor photobleaching FRET); the recorded values were converted into FRET efficiency (17). Fluorescence recovery after photobleaching (FRAP) was also recorded on a Zeiss LSM510 confocal laser scanning microscope.…”

“…The plasmid encoding shRNA was also transfected using Lipofectamine Plus. Uptake of [ 3 H]GABA was determined as described in detail elsewhere (17). In brief, transiently transfected cells (3 ϫ 10 5 /well) were seeded into 24-well dishes 24 h after transfection; after 12 h, the cells were incubated in the presence of 20 M [ 3 H]GABA (specific activity ϳ70 cpm/pmol) for 3 min; nonspecific uptake was defined as the amount of cell associated radioactivity determined in the presence of 10 M tiagabine.…”

“…Accordingly, the transporter of interest must be trapped in the ER: if the leucine repeat in transmembrane segment 2 of GAT1 is disrupted by substitution with alanines, the resulting GAT1-L2A is defective in oligomerization and retained in the ER (12). This mutant still interacts with Sec24D (17). We introduced an analogous mutation in the leucine heptad repeat of the DAT; disruption of the leucine heptad repeat of DAT also leads to retention of the resulting mutants in the ER (13) and abolishes oligomer formation (not shown).…”

“…We also located a sequence element composed of 10 amino acids (Leu 553 -Gln 572 ) in the GAT1 carboxyl terminus, which mediated an interaction with Sec24D (17). We surmised that the hydrophobic cluster 569 VMI 571 conformed to a di-hydrophobic ER export (7).…”