Sorting of the vesicular GABA transporter to functional vesicle pools by an atypical dileucine-like motif

Abstract

Increasing evidence indicates that individual synaptic vesicle proteins may use different signals, endocytic adaptors, and trafficking pathways for sorting to distinct pools of synaptic vesicles. Here, we report the identification of a unique amino acid motif in the vesicular GABA transporter (VGAT) that controls its synaptic localization and activity-dependent recycling. Mutational analysis of this atypical dileucine-like motif in rat VGAT indicates that the transporter recycles by interacting with the clathrin adaptor protein AP-2. However, mutation of a single acidic residue upstream of the dileucine-like motif leads to a shift to an AP-3-dependent trafficking pathway that preferentially targets the transporter to the readily releasable and recycling pool of vesicles. Real-time imaging with a VGAT-pHluorin fusion provides a useful approach to explore how unique sorting sequences target individual proteins to synaptic vesicles with distinct functional properties.

Figure 1.

Mutagenesis screen of putative VGAT trafficking motifs. A, Alignment of putative targeting sequences in VGAT with similar sequences from other vesicular neurotransmitter transporters (bold, underlined). The dileucine-like sequences are in bold (Tan et al., 1998; Voglmaier et al., 2006). B, Mutations as indicated were used to map amino acid residues involved in VGAT trafficking. C, Mutation of F₄₄ in a putative atypical dileucine motif to alanine (F₄₄A/AA) results in increased VGAT localization at the plasma membrane (bottom), whereas mutation of a more typical dileucine pair (I₆₂L₆₃/AA) or a polyproline domain (P₉₁P₉₄/AA) does not affect VGAT localization (middle). HEK cells expressing the indicated VGAT mutants were fixed, permeabilized and stained with rabbit antibody to VGAT and mouse monoclonal antibody to transferrin receptor (TfR), followed by appropriate secondary antibodies. Acquisition parameters were identical for all conditions. Scale bar, 10 μm.

Figure 2.

Development of a pHluorin-based reporter of VGAT vesicle recycling. A, Schematic representation of VGAT-pH depicts pHluorin inserted at the lumenal C-terminus of rat VGAT. B, VGAT-pH fluorescence colocalizes with VGLUT1 antibody staining at varicosities in transfected hippocampal neurons. Scale bar, 10 μm. C, Time-lapse images show the fluorescence change of VGAT-pH in response to neural activity. After onset of a 10 Hz 60 s stimulus, exocytosis of VGAT-pH results in a rapid increase in fluorescence (15, 30, and 60 s), followed by fluorescence decay after the end of stimulation (75, 120 and 300 s) as the vesicles undergo endocytosis and reacidification. Color scale is shown to the right. Scale bar, 2 μm. D, Fluorescence intensity, normalized to baseline (ΔF/F₀), of VGAT-pH (black) increases during stimulation (bar) and decays with an exponential time course after termination of the stimulus (τ = 33.03 ± 1.79 s), consistent with exocytosis followed by endocytosis. No change in fluorescence of VGAT-pH occurs in the absence of calcium (red). E, There is no difference in the rate of FM4-64 destaining between boutons from untransfected (red) and transfected (black) neurons. F, Kinetics of fluorescence changes are similar for VGAT-pH transfected in hippocampal (black, τ = 33.03 ± 1.79 s) and striatal (gray, τ = 30.55 ± 2.98 s) cultures. Data in D–F are means ± SEM of at least 20 boutons per coverslip (cs) from 6 to 9 cs from at least three independent cultures.

Figure 3.

A single dileucine-like motif controls VGAT-pH synaptic targeting and recycling. A, Mutation of F₄₄ to alanine (F₄₄A/AA, red) and of E₃₉E₄₀ to glycine (E₃₉E₄₀/GG, blue) slows VGAT-pH endocytosis after stimulation (WT: black, τ = 33.03 ± 1.79 s, FA/AA: τ = 86.88 ± 7.37 s, EE/GG: τ = 61.84 ± 2.84 s). B, Left and middle, The fluorescence of both WT VGAT-pH and the FA/AA mutant expressed in striatal and hippocampal neurons is largely quenched before stimulation (0 s). Mutation of F₄₄A₄₅ to GG results in an altered distribution throughout the processes (GG). Upon stimulation (60 s), WT and AA exhibit fluorescence increases consistent with exocytosis, but maintain a punctate localization. Mutation of FA to GG abolishes synaptic targeting and response to stimulation. Right, the analogous mutations in VGLUT1-pH in hippocampal neurons show that VGAT-pH and VGLUT1-pH GG mutants display differences in cell surface localization (GG, 0 s) and response to stimulation (GG, 0 vs 60 s). C, Fluorescence of WT VGAT-pH is quenched at resting conditions, and thus not sensitive to acid quenching by pH 5.5 Tyrode's solution added to the outside of cells (top). Mutation of residues E₃₉, E₄₀ and F₄₄ renders VGAT-pH sensitive to acid quenching (4A, bottom), similar to FA/GG (middle). D, The surface fraction of WT VGAT-pH and D/G and EE/GG mutants are not significantly different before stimulation (WT, 2.6 ± 0.5%; D/G, 2.9 ± 0.3%; EE/GG, 4.1 ± 0.4%). However, the surface fraction of EE/GG is significantly higher after stimulation (WT, 2.7 ± 0.4%; D/G, 3.0 ± 0.5%; EE/GG, 8.7 ± 1.0%). FA/AA exhibits higher cell-surface expression both before (10.3 ± 0.7%) and after (19.3 ± 0.8%) stimulation. Solid and hatched bars represent fraction of surface fluorescence at steady-state and after endocytosis is allowed to proceed for 4 min after stimulation, respectively. Results are mean ± SEM of ΔF/F₀ normalized to total fluorescence (NH₄Cl) of at least 20 boutons per cs from 8 to 15 cs from 3 to 5 independent cultures. ***p < 0.001, one-way ANOVA.

Figure 4.

VGAT interacts with clathrin adaptor proteins through its dileucine-like motif. A, A GST fusion of the WT VGAT N-terminus pulls down AP-2 from rat brain lysates, but mutation of the dileucine-like motif disrupts the interaction. The D/G mutant interacts more strongly with AP-3, whereas none of the mutations tested affects interaction of VGAT with AP-1. Bound proteins were detected by immunoblotting with antibodies against AP-1 (anti-adaptin γ), AP-2 (anti-adaptin α), and AP-3 (anti-β-NAP). Top panels show representative immunoblots, bottom show the averaged quantification of band intensity from at least three independent experiments. *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA. B, No binding to AP180 or stonin 2 is detected with the same pull down experiments described in A and immunoblotting with antibodies against AP180 and stonin 2. C, AP-2 protein, but not AP-1or AP-3, is specifically depleted by lentiviral expression of AP-2μ2 shRNA, as shown by immunoblot analysis of hippocampal neuron extracts (left). Right, quantified band intensity, averaged from 2 independent experiments, ***p < 0.0001, one-way ANOVA. D, Depletion of AP-2 slows endocytosis of VGAT-pH after stimulation, whereas expression of the shRNA-resistant μ2 rescues this effect (control τ = 52.46 ± 5.65 s, AP-2KD τ = 129.73 ± 9.21 s, rescue τ = 60.71 ± 8.36 s). Data are means ± SEM of ΔF/F₀ normalized to total fluorescence from at least 30 boutons per cs from 7 to 14 cs from at least two independent cultures. Bottom, In neurons depleted of AP-2, a larger fraction of VGAT-pH remains on the cell surface after stimulation and recovery, which is reversed by expression of the shRNA-resistant μ2 (control 3.6 ± 0.8%, AP2-KD 16.8 ± 2.6%, rescue 5.6 ± 0.3%), ***p < 0.001, one-way ANOVA.

Figure 5.

Mutation of D₃₈ to G alters VGAT recycling kinetics during stimulation. A, Upon stimulation, fluorescence of D/G VGAT-pH (green, ΔF/F₀ normalized to total fluorescence) rises faster and peaks higher than WT (black) (WT peak: 0.5050 ± 0.0012, D/G peak: 0.6123 ± 0.0007, p < 0.0001 by two-tailed, unpaired t test). B, BFA has no effect on the recycling of WT VGAT-pH (hatched black bar), but reduces the peak fluorescence of D/G VGAT-pH (hatched green bar), **p = 0.0012 by two-tailed, unpaired t test. C, D/G VGAT-pH (green) exhibits a faster initial rate of exocytosis (τ = 13.6 ± 0.1 s) compared with WT VGAT-pH (black, τ = 22.3 ± 0.2 s). The proportion of D/G that undergoes exocytosis (0.688 ± 0.003) is also significantly increased compared with WT (0.593 ± 0.001). Neither FA/AA (red) nor EE/GG (blue) mutation significantly alters the rate of VGAT-pH exocytosis (FA/AA, τ = 24.9 ± 0.2 s; EE/GG, τ = 24.7 ± 0.3 s) or the size of the recycling pool. D, The rate of FM5-95 destaining is not significantly different between boutons transfected with WT and D/G VGAT-pH. Data are means ± SEM of ΔF/F₀ normalized to total fluorescence from at least 20 boutons per cs from 6 to 17 cs from at least three independent cultures.

Figure 6.

Increased D/G VGAT-pH in the RRP is mediated by AP-3. A, Exocytosis from the RRP is evoked using a stimulus of 20 action potentials at 100 Hz. Inset: the fraction of D/G VGAT-pH (green) released by RRP stimulation (7.14 ± 0.04%) is ∼50% larger than WT VGAT-pH (4.69 ± 0.05%, black), ***p < 0.0001 by two-tailed, unpaired t test. B, Exocytosis from the RRP is evoked by application of Tyrode's solution containing 300 mm sucrose in the presence of 1 μm bafilomycin to prevent reacidification of internalized vesicles. The fraction of D/G VGAT-pH (green) released (7.64 ± 0.48%) is ∼40% larger than WT VGAT-pH (5.48 ± 0.51%, black), **p < 0.01 by two-tailed, unpaired t test. C, AP-3δ protein, but not AP-1γ, or AP-2α, is specifically depleted by lentiviral expression of AP-3δ1 shRNA, as shown by immunoblot analysis of hippocampal neuron extracts (left). Right, Quantified band intensity, averaged from two independent experiments, ***p < 0.0001, one-way ANOVA. D, AP-3 knockdown (open symbols) does not affect the fraction of WT VGAT-pH in the RRP. Conversely, AP-3 knockdown decreases the fraction of D/G VGAT-pH in the RRP to WT levels. E, The fraction of WT VGAT in the RRP is insensitive to treatment with BFA. In contrast, the proportion of D/G VGAT-pH in the RRP reverts to WT levels after BFA treatment. F, Treatment of AP-3 KD neurons with BFA has no additional effect on the fraction of D/G VGAT in the RRP. G, Similar results are observed with an alternate stimulus of 30 Hz 3 s to release the RRP. Expression of shRNA resistant δ1 rescues the AP-3 KD-mediated effect. Fluorescence changes upon RRP stimulation were measured in the presence (A, B) or absence (D–G) of bafilomycin. Data in A, B, D–G are means ± SEM of ΔF/F₀ normalized to total fluorescence from at least 20 boutons per cs from 6 to 19 cs from at least two independent cultures.