The activity of serotonergic systems depends on the reuptake of extracellular serotonin via its plasma membrane serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT), a member of the Na+/Cl-dependent solute carrier 6 family. SERT is finely regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins. The majority of previously identified SERT partners that control its functional activity are soluble proteins, which bind to its intracellular domains. SERT also interacts with transmembrane proteins, but its association with other plasma membrane transporters remains to be established. Using a proteomics strategy, we show that SERT associates with ASCT2 (alanine–serine–cysteine–threonine 2), a member of the solute carrier 1 family co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane. Co-expression of ASCT2 with SERT in HEK (human embryonic kidney)-293 cells affects glycosylation and cell-surface localization of SERT with a concomitant reduction in its 5-HT uptake activity. Conversely, depletion of cellular ASCT2 by RNAi enhances 5-HT uptake in both HEK-293 cells and primary cultured mesencephalon neurons. Mimicking the effect of ASCT2 down-regulation, treatment of HEK-293 cells and neurons with the ASCT2 inhibitor D-threonine also increases 5-HT uptake. Moreover, D-threonine does not enhance further the maximal velocity of 5-HT uptake in cells depleted of ASCT2. Collectively, these findings provide evidence for a complex assembly involving SERT and a member of another solute carrier family, which strongly influences the subcellular distribution of SERT and the reuptake of 5-HT.

INTRODUCTION

The homoeostasis of serotonergic transmission is critically dependent on 5-HT [5-HT (5-hydroxytryptamine), also known as serotonin] reuptake into presynaptic neurons via its plasma membrane transporter SERT, a member of the SLC6 (solute carrier 6) family of Na+/Cl-dependent transporters that also includes transporters for the biogenic amines noradrenaline (norepinephrine) and dopamine, and transporters for the amino acid transmitters GABA (γ-aminobutyric acid) and glycine [1,2]. The mechanisms controlling SERT functional activity raise particular interest, as it represents the primary target for several widely prescribed antidepressants such as tricyclics and SSRIs (selective serotonin reuptake inhibitors) and for psychostimulants such as cocaine and amphetamine derivatives [35]. Moreover, deregulation of SERT has been associated with numerous psychiatric disorders, including anxiety, depression, impulsive behaviour and alcoholism [1,2].

Multiple mechanisms have been implicated in the regulation of SERT plasma membrane expression, trafficking and 5-HT uptake activity. These include post-translational modifications, such as glycosylation [68], nitrosylation [9] and phosphorylation at multiple sites [10,11]. SERT's phosphorylation status is finely modulated by a complex network of protein kinases including PKC (protein kinase C) [1214], PKG (protein kinase G) [15,16], p38 MAPK (mitogen-activated kinase) [17], Akt [18] and as yet unidentified protein kinases [19]. SERT phosphorylation is also regulated by protein phosphatases, such as PP2A (protein phosphatase 2A) [20] and the Ca2+/calmodulin-dependent phosphatase calcineurin [21]. In addition, SERT interacts with numerous intracellular proteins, which control its subcellular distribution and intrinsic transport activity [10,11]. The emerging view is that the physiological functions of SERT are dynamically modulated by a complex interplay between post-translational modifications of SERT and its physical association with protein partners. For instance, association of SERT with the PKC substrate MacMARCKS (macrophage myristoylated alanine-rich C-kinase substrate) affects PKC-dependent down-regulation of SERT transport activity [22]. Moreover, Ca2+ and calcineurin phosphatase activity enhance SERT association with calcineurin, which promotes SERT dephosphorylation and likewise prevents the down-regulation of SERT function caused by PKC activation [21].

The majority of SERT partners characterized to date have been identified by means of two-hybrid screens, most often based on their previously described interactions with other SLC6 family transporters [2229] or by proteomics strategies combining AP (affinity purification) of SERT-associated proteins and their identification by MS (AP–MS strategies) [21,3033]. These previously employed strategies primarily targeted interactions with the cytoplasmic loops and terminal segments of the transporter and identified not only soluble partners [2123,25,28,30,31,33], but also transmembrane proteins, including two SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, namely syntaxin 1A [34,35] and VAMP2 (vesicle-associated membrane protein 2) [36], SCAMP2 (secretory carrier membrane protein 2) [26], integrin αIIbβ3 [24] and membrane glycoprotein M6B [29]. It is likely that SERT recruits additional partners via multiple discontinuous interfaces, involving not only cytosolic domains, but also transmembrane regions (TMs). For instance, like many transport proteins, SERT constitutively forms oligomers (most likely tetramers) and assembly of SERT oligomers occurs via TM11–TM12 and TM1–TM2 interactions [37,38]. Whether SERT associates with plasma membrane transporters belonging to different solute carrier families remains to be established.

In the present study, using a complementary AP–MS strategy, we found that SERT interacts with the neutral amino acid transporter ASCT2 (alanine–serine–cysteine–threonine 2), a member of the SLC1 family of transporters specific for the transport of small neutral amino acids, such as glutamine, alanine, serine and cysteine [39], both in HEK (human embryonic kidney)-293 cells and in mouse brain. These experiments were complemented by a series of functional studies, which revealed that ASCT2 expression prevents SERT maturation and plasma membrane localization. These studies also showed that SERT-operated 5-HT uptake is regulated by ASCT2 expression and its amino acid transport activity in both HEK-293 cells and primary mesencephalon neurons.

EXPERIMENTAL

Materials

[3H]5-HT creatine sulfate (95 Ci/mmol), [3H]dihydroxypheny-lethylamine dopamine (38.7 Ci/mmol), [3H]noradrenaline hydrochloride (14.9 Ci/mmol), [3H]alanine (84.1 Ci/mmol) and [3H]glutamine (50.3 Ci/mmol) were purchased from PerkinElmer. Oligonucleotides were from Eurogentec. Cell culture media and antibiotics were from Invitrogen. All chemicals were from Sigma–Aldrich.

The plasmids encoding YFP-tagged human SERT (pEYFP/SERT), dopamine transporter (DAT) (pEYFP/DAT) and noradrenaline transporter (NET) (pEYFP/NET) were kindly provided by Dr Michael Freissmuth (Medical University of Vienna, Vienna, Austria). The FLAG-tagged human ASCT2 cDNA, cloned in the pCDNA5 plasmid, was obtained from the human ORFeome platform (IGMM, Montpellier, France). The cDNA encoding mouse ASCT2 (in pFLCII), purchased from Source Bioscience, was cloned in pCDNA3.1+ plasmid using NheI/NotI cloning sites.

Mouse monoclonal anti-GFP antibody (mixture of clones 7.1 and 13.1) was from Roche Diagnostics, polyclonal anti-FLAG antibody was from Sigma–Aldrich, polyclonal anti-SERT antibody used for immunodetection of native SERT was from Immunostar, polyclonal anti-SERT antibody used for immunoprecipitation of native SERT, polyclonal antibody against mouse ASCT2 used for immunodetection and immunoprecipitation of native ASCT2 and polyclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody were from Santa Cruz Biotechnology. Polyclonal antibody against human ASCT2 used for detection of ASCT2 in HEK-293 cells was from Cell Signaling Technology.

Cell cultures and transfections

HEK-293 cells were grown at 37°C in a 5% CO2 humidified atmosphere, in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) dialysed FBS. Cells were used after six to 12 passages and transfected by electroporation using the Bio-Rad Laboratories Gene Pulser X cell. Each transfection sample contained 5×106 cells and 0.25 μg of SERT, DAT, NET or ASCT2 constructs, and empty pRK5 vector for a total of 5 μg of DNA. Electroporated cells were plated on polyornithine-coated 96-well plates for uptake experiments, or 100-mm-diameter dishes for co-immunoprecipitation experiments, or 12-mm glass slides placed in 24-well culture dishes (105 cells/well or slide and 5×106 cells/dish respectively). Cells were starved of serum 24 h after transfection and experiments were carried out 48 h after transfection. Immunofluorescence experiments indicated that >50% of the cells were transfected with SERT, DAT, NET or ASCT2 constructs.

Primary cultures of mesencephalon neurons were prepared from 13-day-old mouse embryos, as described previously [40]. Briefly, cells were plated on polyornithine-coated 12- or 24-well culture dishes (106 cells/well and 5×105 cells/well respectively) in serum-free medium consisting of DMEM/Ham's F12 (1:1, v/v), 33 mM D-glucose, 2 mM L-glutamine, 13 mM NaHCO3 and 5 mM Hepes, pH 7.4, and supplemented with 10% hormone and salt mixture (25 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine, 20 nM progesterone and 30 nM sodium selenite). Cultures were maintained at 37°C in a humidified atmosphere under 5% CO2, and used 8 days after seeding.

ASCT2 silencing in HEK-293 cells and primary neurons

HEK-293 cells (3×106 per condition, six to ten passages) were transfected in suspension with 200 nM of either Slc1a5 pre-designed Mission® siRNAs (siRNA1, 5′-GGCGGCAG-TGTTCATCGCACA-3′, siRNA2, 5′-CAGTGTTCATCGCAC-AACTAA-3′, siRNA3, 5′-AACCCAAAGACAACTCATGTA-3′, Sigma–Aldrich) or Universal Negative Control siRNA (Sigma–Aldrich) and 1 μg of the pEYFP/SERT plasmid using Lipofectamine™ (Invitrogen). Cells were plated on polyornithine-coated 96-well plates (105 cells/well) and medium was changed after 6 h. They were starved of serum 24 h after transfection and used 48 h after transfection. Analysis of ASCT2 expression in siRNA-transfected cells by Western blotting using polyclonal anti-ASCT2 antibody showed that only siRNA1 efficiently silenced ASCT2 expression, whereas siRNA2 partially decreased the ASCT2 level and siRNA3 was ineffective. Therefore siRNA1 was used in further experiments.

Primary mouse mesencephalon neurons grown in 12-well culture plates were infected with lentiviral particles containing an shRNA corresponding to the same sequence as the ASCT2 siRNA1. ASCT2 shRNA and control shRNA cloned into the pLKO.1-puro lentiviral vector were obtained from Sigma–Aldrich. Lentiviruses were produced by the AnIRA Vectorology platform. Neurons were used 72 h after infection. Down-regulation of ASCT2 expression was analysed by qRT-PCR (quantitative reverse transcription–PCR) on a Lightcycler (using primers ASCT2-fw, 5′-TGCTTTCGGGACCTCTTCTA-3′, and ASCT2-rev, 5′-TGATGTGTTTGGCCACACCA-3′) and normalized to the geometric mean of the expression levels of β2-microglobulin and tubulin mRNAs.

Co-immunoprecipitation

For GFP and YFP-tagged SERT immunoprecipitation, proteins from transfected HEK-293 cells (1 mg for Western blotting or 10 mg for proteomics analyses) were solubilized in a buffer containing 50 mM Tris/HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1% CHAPS and a protease inhibitor cocktail (Roche). Samples were incubated with GFP-Trap beads (Chromotek) for 1 h at 4°C. For FLAG immunoprecipitation, solubilized proteins (1 mg) were incubated with 1 μg of anti-FLAG antibody and immune complexes were captured by incubation with Protein A–Sepharose beads (GE Healthcare) for 1 h at 4°C. Beads were washed five times with 50 mM Tris/HCl, pH 7.4, and immunoprecipitated proteins were analysed by MS or Western blotting. For native SERT or native ASCT2 immunoprecipitation, 50 μl of Protein A/G–Sepharose beads (GE Healthcare) were first incubated overnight at 4°C with 14 μg of either anti-SERT (Santa Cruz Biotechnology) or anti-ASCT2 antibody (Santa Cruz Biotechnology). Thereafter, 2 mg of mice brain proteins solubilized in 50 mM Tris/HCl, pH 7.4, 0.5 mM EDTA, 1.3% CHAPS and a protease inhibitor cocktail (Roche) were pre-cleared with 50 μl of naive Protein A/G–Sepharose beads, and incubated overnight at 4°C with antibody-coated beads. Beads were washed five times successively with low-salt and high-salt buffers containing 50 mM Tris/HCl, pH 7.4, and either 0.15 M or 0.5 M NaCl respectively. Immunoprecipitated proteins were analysed by Western blotting.

Protein identification by MS

After immunoprecipitation, protein identification was performed by Fourier-transform tandem MS as described previously [21]. Briefly, immunoprecipitated proteins were separated by SDS/PAGE, digested in-gel with trypsin and systematically identified by nano-LC–FT–MS/MS (nanoflow liquid chromatography coupled to Fourier-transform tandem MS) using a LTQ-Orbitrap-XL (Thermo Fisher Scientific). The raw MS data were analysed using the MaxQuant /Andromeda software (version 1.4.1.2) [41] with a false discovery rate of less than 0.01 for peptides and proteins (decoy database estimation). Andromeda was used to search the top ten per 100 Da peak lists against the human complete proteome set database downloaded on 8 December 2014 (20194 reviewed protein entries from UniProtKB/Swiss-Prot), combined with 248 frequently observed contaminants as well as reversed versions of all sequences. Enzyme specificity was set to trypsin allowing up to two missed cleavages. Considered modifications were cysteine carbamidomethylation as a fixed modification and protein N-terminal acetylation, and oxidation of methionine as variable modifications. Peptide identification was based on a search with a mass deviation of the precursor ion up to 7 p.p.m. after internal recalibration, and a mass deviation of the fragments up to 0.5 Da. Protein abundances across the different conditions were estimated by calculating the iBAQ (intensity-based absolute quantification) index, which corresponds to the sum of the extracted ion intensities of all identified peptides per protein, normalized by the number of theoretically observable peptides [42].

Western blotting

Proteins resolved by SDS/10% PAGE were transferred electrophoretically on to nitrocellulose membranes (Hybond-C; GE Healthcare). Membranes were incubated in blocking buffer (50 mM Tris/HCl, pH 7.5, 200 mM NaCl, 0.1% Tween 20 and 5% non-fat dried skimmed milk powder) for 1 h at room temperature, and overnight with primary antibodies (mouse anti-GFP 1:500 dilution; rabbit anti-FLAG 1:1000 dilution; rabbit anti-ASCT2 1:200 dilution) in blocking buffer at 4°C. They were washed three times with blocking buffer and incubated with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:5000 dilution; GE Healthcare) for 1 h at room temperature. Immunoreactivity was detected with an ECL method (ECL detection reagent, GE Healthcare).

Immunohistochemistry

Adult Swiss mice were rapidly anaesthetized with pentobarbital (100 mg/kg intraperitoneal injection; Ceva SA) and perfused transcardially with fixative solution containing 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5). Brains were then post-fixed with the same solution for 4 days at 4°C. Sections 50-μm-thick were cut with a vibratome (Leica) and incubated for 2 h in 1% (w/v) BSA and PBS/0.2% Triton X-100, then for 3 days at 4°C with rabbit anti-SERT (1:2500 dilution; Immunostar) and goat anti-ASCT2 (1:100 dilution; Santa Cruz Biotechnology) in 1% (w/v) BSA and PBS/0.2% Triton X-100. Brain sections were washed three times with PBS, incubated for 2 h with biotinylated goat anti-rabbit IgG (1:500 dilution; Dako) in 1% (w/v) BSA and PBS/0.2% Triton X-100, washed again three times with PBS and incubated for 2 h with Alexa Fluor 488-coupled donkey anti-goat IgG (Invitrogen) and Texas Red-coupled avidin D (Vector Laboratories). After three washes with PBS, sections were mounted with Mowiol. Immunofluorescent staining was observed with a Zeiss Axiophot2 microscope equipped with epifluorescence. Images were acquired using the Metamorph software (Molecular Devices) driving a CoolSNAP CCD (charge-coupled device) camera (Photometrics). Post-treatment of images (level correction), annotations and panel composition were performed using Photoshop software (Adobe).

Immunocytochemistry and Apotome fluorescence microscopy

HEK-293 cells expressing YFP-tagged SERT and FLAG-tagged ASCT2 (alone or in combination) were grown on glass coverslips for 48 h after transfection and fixed in 4% (w/v) paraformaldehyde in PBS for 10 min. They were washed twice with 0.1 M glycine and incubated with PBS supplemented with 0.05% Triton X-100 and 10% goat serum for 30 min. They were then successively incubated with primary antibodies (mouse anti-GFP, 1:1000 dilution, and/or rabbit anti-FLAG, 1:1000 dilution) and with secondary antibodies (Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 594-conjugated anti-rabbit, 1:1000 dilution). Coverslips were mounted with Mowiol. Fluorescent pictures were acquired using an AxioImager Z1 microscope equipped with an AxioCam MRm camera, Plan Apochromat [×40, NA (numerical aperture) 0.95; ×63, NA 1.4] objectives and the Apotome Slider system with an H1 transmission grid (Carl Zeiss), using AxioVision software (Carl Zeiss). Post-treatment of images (level correction), annotations and panel composition were performed using Photoshop software.

Monoamine and amino acid uptake assays

HEK-293 cells grown in 96-well culture dishes were washed in Hepes buffer (150 mM NaCl, 4.2 mM KCl, 0.9 mM CaCl2, 0.5 mM MgSO4, 5 mM glucose and 10 mM Hepes, pH 7.4) and incubated for 10 min at 37°C in Hepes buffer containing 100 μM pargyline and 100 μM ascorbic acid. 5-HT uptake was initiated by the addition of [3H]5-HT (20 nM) and increasing concentrations of non-radiolabelled 5-HT ranging from 0.1 to 50 μM (for 10 min at 37°C) and terminated by three washes with ice-cold Hepes buffer containing 10 μM fluoxetine, followed by cell lysis in 1% (w/v) SDS. Non-specific 5-HT uptake was determined in the presence of 10 μM fluoxetine. For 5-HT uptake into cultured mesencephalon neurons, cultures were plated in 12-well culture dishes, pre-treated as indicated above, and uptake was initiated by the addition of 100 nM [3H]5-HT. Non-specific 5-HT uptake was determined in the presence of 50 nM fluoxetine. The radioactivity incorporated into cells was determined by scintillation counting.

Dopamine and noradrenaline uptake was performed in HEK-293 cells using a procedure similar to that used for [3H]5-HT uptake. Uptake was initiated by the addition of [3H]dopamine (20 nM) or [3H]noradrenaline (20 nM) and increasing concentrations of non-radiolabelled dopamine or noradrenaline ranging from 0.5 to 30 μM. Determination of non-specific dopamine and noradrenaline uptake was conducted in the presence of 5 μM GBR12909 and 10 μM imipramine respectively. Note that GBR12909 and imipramine did not affect [3H]dopamine and [3H]noradrenaline uptake respectively in HEK-293 cells transfected with the empty construct.

Alanine and glutamine uptake was performed in HEK-293 cells and mesencephalon neurons using procedures similar to those used for [3H]5-HT uptake in both cell populations. Uptake was initiated by the addition of [3H]alanine or [3H]glutamine (20 nM in neurons, or 600 μM in HEK-293 cells) and increasing concentrations (0.05 to 50 mM) of D-threonine, an inhibitor of neutral amino acid transport via ASCT2.

Biotinylation assay

Biotinylation was performed in transfected HEK-293 cells using the Cell Surface Isolation kit (Pierce), according to the manufacturer's instructions. Briefly, 48 h after transfection, cells were washed in ice-cold PBS (pH 8.0) and incubated with 1 mg/ml sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] under gentle agitation for 15 min at 4°C. After washing, cells were scraped off in 1 ml of lysis buffer and lysates were centrifuged at 8000 g for 10 min. Clarified cell lysates were then incubated overnight at 4°C with 200 μl of neutravidin–agarose beads. The beads were washed three times in lysis buffer and once in 50 mM Tris/HCl, pH 7.4, and proteins retained on the beads were eluted with 100 μl of SDS sampling buffer. The amount of SERT was analysed in lysates and eluates (biotinylated fraction) by Western blotting, using monoclonal anti-GFP antibody.

Statistics

Data analysis and statistics were carried out by using GraphPad Prism 5 software. Data were analysed using Student's t test, except for those illustrated in Figures 3(E) and 4(E), which were analysed by one-way ANOVA followed by a Newman–Keuls test and Dunnett's test respectively.

RESULTS AND DISCUSSION

Association of SERT with the neutral amino acid transporter ASCT2

To identify partners recruited by the full-length SERT protein, including proteins that bind to the transporter via discontinuous interfaces not only located in cytosolic domains, but also in transmembrane regions, we employed a proteomics strategy combining the purification of SERT-associated proteins by co-immunoprecipitation and their identification by high-resolution tandem MS. Owing to the low density of SERT in mammalian brain and the lack of an antibody permitting immunoprecipitation yields compatible with MS analysis, we used SERT tagged N-terminally with YFP expressed in HEK-293 cells as bait. This approach showed unequivocal efficiency in identifying protein partners of membrane receptors, such as the 5-HT6 receptor and melatonin receptors [43,44]. Moreover, we showed previously that YFP-tagged SERT is expressed at the plasma membrane and is functional [31]. Immunoprecipitation was performed by using the GFP-Trap technology based on a llama single-chain antibody, which allows efficient purification of proteins fused to GFP (or YFP) and their interacting partners in a one-step procedure, with a limited amount of contaminant proteins in immunoprecipitates [45]. Control immunoprecipitations were performed from cells transfected with a plasmid encoding GFP alone.

Analysis of immunoprecipitated proteins by SDS/PAGE revealed the presence of proteins recruited by SERT and that are not detectable in control immunoprecipitates (Figure 1A). Correspondingly, systematic analysis by nano-LC–MS/MS (nanoflow liquid chromatography coupled with tandem MS) of gel lanes reproducibly identified 12 proteins (with at least two peptides) that co-immunoprecipitated with YFP–SERT but were absent from control immunoprecipitates in two biological replicates (Table 1).

SERT recruits ASCT2 in HEK-293 cells

Figure 1
SERT recruits ASCT2 in HEK-293 cells

(A) HEK-293 cells were transfected with plasmids encoding either GFP or YFP-tagged SERT. After cell lysis, solubilized protein extracts were incubated with GFP-Trap beads. Proteins retained by affinity were resolved by SDS/PAGE and stained with colloidal Coomassie Blue. A representative gel is shown. (B and C) HEK-293 cells were co-transfected with plasmids encoding either GFP or YFP-tagged SERT or FLAG-tagged ASCT2, or co-transfected with YFP-tagged SERT and FLAG-tagged ASCT2. Proteins were immunoprecipitated with either GFP-Trap beads (B) or a monoclonal anti-FLAG antibody conjugated to Sepharose beads (C). SERT and ASCT2 levels in inputs and immunoprecipitates were analysed by Western blotting using monoclonal anti-GFP and polyclonal anti-FLAG antibody respectively. Inputs represent 2% of the total protein amount used for immunoprecipitations. The arrows show the position of immunoreactive bands corresponding to YFP–SERT (upper panels) and FLAG–ASCT2 (lower panels), respectively. (D) HEK-293 cells were transfected with the plasmid encoding YFP-tagged SERT alone or in combination with the plasmid encoding FLAG-tagged ASCT2. Cell lysates were treated or not with PNGase F (1 unit) for 1 h at 37°C and analysed by Western blotting using monoclonal anti-GFP antibody. The data in (B), (C) and (D) are representative of three independent experiments. Mw, molecular mass (indicated in kDa). IP, immunoprecipitation.

Figure 1
SERT recruits ASCT2 in HEK-293 cells

(A) HEK-293 cells were transfected with plasmids encoding either GFP or YFP-tagged SERT. After cell lysis, solubilized protein extracts were incubated with GFP-Trap beads. Proteins retained by affinity were resolved by SDS/PAGE and stained with colloidal Coomassie Blue. A representative gel is shown. (B and C) HEK-293 cells were co-transfected with plasmids encoding either GFP or YFP-tagged SERT or FLAG-tagged ASCT2, or co-transfected with YFP-tagged SERT and FLAG-tagged ASCT2. Proteins were immunoprecipitated with either GFP-Trap beads (B) or a monoclonal anti-FLAG antibody conjugated to Sepharose beads (C). SERT and ASCT2 levels in inputs and immunoprecipitates were analysed by Western blotting using monoclonal anti-GFP and polyclonal anti-FLAG antibody respectively. Inputs represent 2% of the total protein amount used for immunoprecipitations. The arrows show the position of immunoreactive bands corresponding to YFP–SERT (upper panels) and FLAG–ASCT2 (lower panels), respectively. (D) HEK-293 cells were transfected with the plasmid encoding YFP-tagged SERT alone or in combination with the plasmid encoding FLAG-tagged ASCT2. Cell lysates were treated or not with PNGase F (1 unit) for 1 h at 37°C and analysed by Western blotting using monoclonal anti-GFP antibody. The data in (B), (C) and (D) are representative of three independent experiments. Mw, molecular mass (indicated in kDa). IP, immunoprecipitation.

Table 1
List of candidate protein partners of SERT identified by MS/MS

For each protein, the number of unique peptides identified by MS/MS, the corresponding sequence coverage, its molecular mass and iBAQ index are indicated.

Protein IDUniProt accession numberApproved protein nameUnique peptidesCoverage (%)Molecular massiBAQ (×106)Soluble proteinsMembrane proteins
SC6A4 P31645 Sodium-dependent serotonin transporter (SERT) 17 28.4 70.3 478.8  
AAAT Q15758 Neutral amino acid transporter B0 (ASCT2) 13 32.9 56.6 18.4  
PGRC1 O00264 Membrane-associated progesterone receptor component 1 (PGRMC1) 34.9 21.7 17.0  
SCAM3 O14828 Secretory carrier-associated membrane protein 3 (SCAMP3) 17.3 38.3 16.7  
DNJA1 P31689 DnaJ homologue subfamily A member 1 24.7 44.9 12.8  
2AAA P30153 Serine/threonine protein phosphatase 2A 65 kDa regulatory subunit A α isoform (PP2A) 11 24.4 65.3 9.8  
TMCO1 Q9UM00 Transmembrane and coiled-coil domain-containing protein 1 15.4 21.2 7.5  
TMEDA P49755 Transmembrane emp24 domain-containing protein 10 (Tmp21) 20.5 25.0 6.1  
PSMD3 O43242 26S proteasome non-ATPase regulatory subunit 3 14.8 61.0 3.4  
4F2 P08195 4F2 cell-surface antigen heavy chain (4F2hc, CD98hc) 18.6 68.0 1.7  
S12A2 P55011 Solute carrier family 12 member 2 (NKCC1) 9.0 131.5 0.7  
NICA Q92542 Nicastrin 18.2 78.4 0.7  
HYOU1 Q9Y4L1 Hypoxia up-regulated protein 1 (HYOU1) 8.3 111.3 0.3  
Protein IDUniProt accession numberApproved protein nameUnique peptidesCoverage (%)Molecular massiBAQ (×106)Soluble proteinsMembrane proteins
SC6A4 P31645 Sodium-dependent serotonin transporter (SERT) 17 28.4 70.3 478.8  
AAAT Q15758 Neutral amino acid transporter B0 (ASCT2) 13 32.9 56.6 18.4  
PGRC1 O00264 Membrane-associated progesterone receptor component 1 (PGRMC1) 34.9 21.7 17.0  
SCAM3 O14828 Secretory carrier-associated membrane protein 3 (SCAMP3) 17.3 38.3 16.7  
DNJA1 P31689 DnaJ homologue subfamily A member 1 24.7 44.9 12.8  
2AAA P30153 Serine/threonine protein phosphatase 2A 65 kDa regulatory subunit A α isoform (PP2A) 11 24.4 65.3 9.8  
TMCO1 Q9UM00 Transmembrane and coiled-coil domain-containing protein 1 15.4 21.2 7.5  
TMEDA P49755 Transmembrane emp24 domain-containing protein 10 (Tmp21) 20.5 25.0 6.1  
PSMD3 O43242 26S proteasome non-ATPase regulatory subunit 3 14.8 61.0 3.4  
4F2 P08195 4F2 cell-surface antigen heavy chain (4F2hc, CD98hc) 18.6 68.0 1.7  
S12A2 P55011 Solute carrier family 12 member 2 (NKCC1) 9.0 131.5 0.7  
NICA Q92542 Nicastrin 18.2 78.4 0.7  
HYOU1 Q9Y4L1 Hypoxia up-regulated protein 1 (HYOU1) 8.3 111.3 0.3  

Identified proteins comprise a majority of integral membrane proteins. These include plasma membrane transporters such as the neutral amino acid transporter ASCT2 and SLC12 member 2 (also called Na+–K+–2Cl co-transporter isoform 1 or NKCC1), a cation–chloride co-transporter important for maintaining proper ionic balance and cell volume. Interestingly, SERT also recruited 4F2hc (4F2 cell-surface antigen heavy chain, also called CD98hc), a multifunctional glycoprotein with a single transmembrane domain, which functions as a chaperone within a large complex of membrane proteins comprising amino acid transporters such as ASCT2 and monocarboxylate transporters [46]. The SERT-associated proteins also include two components of the presenilin complex, namely Tmp21 (transmembrane emp24 domain-containing protein 10), a protein involved in vesicular protein trafficking and that regulates γ-secretase activity [47], and nicastrin [48], a protein that cleaves integral membrane proteins, such as Notch receptors and amyloid precursor protein and plays essential roles in the regulation of short- and long-term synaptic plasticity [49]. Moreover, SERT associates with transmembrane and coiled-coil domain-containing protein 1, a protein involved in ER (endoplasmic reticulum) organization [50], and PGRMC1 (membrane-associated progesterone receptor component 1), one component of the membrane progesterone receptor α. PGRMC1 forms a complex with DCC (deleted in colorectal cancer), the receptor for the axonal guidance cue Netrin1, and might be required for the proper guidance of axons [51]. Finally, SERT recruits SCAMP3, a protein closely related to SCAMP2, a previously identified SERT partner thanks to a two-hybrid screen [26]. Although SCAMP2 did not pass our filters for selecting candidate SERT partners, SCAMP2 was specifically and reproducibly identified in SERT immunoprecipitates, but with only one peptide.

In addition to transmembrane proteins, our proteomic screen identified four soluble proteins (Table 1). These include the regulatory subunit A of PP2A. This contrasts with previous findings, which defined PP2Ac (catalytic subunit of PP2A) as the first SERT-interacting protein [20]. Notably, these studies also showed that the SERT–PP2Ac interaction is dynamically regulated by 5-HT and SERT activity: SERT–PP2Ac is stabilized in the presence of extracellular 5-HT, whereas less active or inactive SERT pools are relatively deficient in SERT–PP2Ac complexes [10,20]. This might explain why we did not detect PP2Ac in SERT immunoprecipitates obtained from cells not exposed to 5-HT. Taken together, these findings suggest that SERT constitutively interacts with the PP2A regulatory subunit, whereas PP2Ac might only be recruited by activated SERT in cells exposed to 5-HT. In addition, we identified in SERT immunoprecipitates two chaperones, namely HYOU1 (hypoxia up-regulated protein 1), a member of the HSP (heat-shock protein) 70 family thought to play an important role in protein folding and secretion in the ER and DnaJ homologue subfamily A member 1, a co-chaperone for HSC70 (heat-shock cognate 70) (Table 1). These results are reminiscent of a recent study, which showed that SERT associates with HSP70-1A and HSP90β [52]. Both take turns to assist in the folding of SERT and thus determine its availability for ER export. Together, these observations suggest that a complex network of chaperones can bind to SERT to assist its folding and ER export once it has adopted a properly folded and stable conformation. The last protein identified (with two peptides) in the SERT interactome is the non-ATPase regulatory subunit 3 of 26S proteasome (Table 1). Rab4 is another previously described SERT-interacting protein [23] that was reproducibly identified in SERT immunoprecipitates but, as for SCAMP2, with only one peptide. Two other ubiquitously expressed partners of SERT (MacMARCKS and Sec 23A) were identified in only one biological replicate, whereas the other ones were not detected by the present screen. This points out the discrepancies between the various approaches used to identify SERT-associated proteins, based on different biological systems for partner purification and focusing on interactions with a specific cytosolic domain (two-hybrid screens and previous AP–MS strategies) or the entire SERT protein (the present study). Consequently, combining those procedures is necessary to achieve a comprehensive overview of the SERT interactome.

An estimation of SERT-associated protein abundance thanks to the calculation of their iBAQ index [42] showed enrichment in ASCT2, compared with the majority of identified partners (Table 1). This observation is consistent with the larger number of trypsic peptides of ASCT2 identified by tandem MS, compared with the other characterized partners, which was in the same range as the number of peptides found for the bait protein (SERT).

ASCT2 is an obligate amino acid exchanger, which has a high affinity for L-alanine, L-serine, L-threonine, L-aspargine and L-glutamine [39]. It is expressed in many tissues, including the brain, where it has been detected in both neurons and astrocytes [53]. It has been proposed that ASCT2 mediates efflux of glutamine from astrocytes, a process critical for recycling synaptically released glutamate through the glutamine/glutamate cycle [54]. ASCT2 might also modulate glutamatergic transmission by contributing to the astrocytic uptake of D-serine, an endogenous co-agonist of glutamatergic NMDA (N-methyl-D-aspartate) receptors [55,56]. In contrast, the role of ASCT2 in serotonergic transmission remained unexplored.

Corroborating the results of our proteomics screen, which suggest a strong interaction between both transporter proteins, co-immunoprecipitation experiments followed by Western blotting showed that FLAG-tagged ASCT2 co-immunoprecipitated with YFP-tagged SERT expressed in HEK-293 cells (Figure 1B). Reciprocally, substantial amounts of YFP-tagged SERT were co-immunoprecipitated with FLAG-tagged ASCT2 (Figure 1C). We next examined possible co-expression of both proteins in serotoninergic neurons by immunohistochemistry using anti-SERT and anti-ASCT2 antibodies that were employed previously to detect mouse SERT and ASCT2 respectively [55,57]. The anti-ASCT2 antibody yielded an immunoreactive band of ∼72 kDa in Western blots obtained from HEK-293 cells expressing recombinant mouse ASCT2 and mouse brain protein extracts, but did not recognize endogenous and recombinant human ASCT2 (Supplementary Figure S1). We found that ASCT2 is expressed in 55% of SERT-positive neurons of the dorsal raphe nucleus (n=178 SERT-positive neurons originating from four different mice counted, Figure 2A), indicating that both protein partners are actually co-expressed in a subpopulation of serotonergic neurons. Supporting further a physical interaction between both proteins in vivo, native ASCT2 was co-immunoprecipitated with native SERT expressed in mouse brain using the anti-SERT antibody (Figure 2B) and, reciprocally, SERT was co-immunoprecipitated with ASCT2 using the anti-mouse ASCT2 antibody (Figure 2C).

SERT and ASCT2 form a complex in vivo

Figure 2
SERT and ASCT2 form a complex in vivo

(A) Immunofluorescent detection of cells positive for SERT and ASCT2 in dorsal raphe nucleus. A representative field from ten slices originating from four mice is shown. Arrows indicate neurons co-expressing SERT and ASCT2, filled arrowheads indicate neurons expressing SERT only and empty arrowheads indicate neurons expressing ASCT2 only. Scale bar, 100 μm. (B and C) Co-immunoprecipitation of native SERT with native ASCT2 expressed in mice brain. Inputs (left-hand panels) represent 1% of the protein amount used for immunoprecipitation. Immunoprecipitations were performed with either the anti-SERT antibody (B) or the anti-mouse ASCT2 antibody (C). Non-immune purified rabbit IgG was used as control. Representative results of three independent experiments are illustrated. Mw, molecular mass (indicated in kDa). IP, immunoprecipitation.

Figure 2
SERT and ASCT2 form a complex in vivo

(A) Immunofluorescent detection of cells positive for SERT and ASCT2 in dorsal raphe nucleus. A representative field from ten slices originating from four mice is shown. Arrows indicate neurons co-expressing SERT and ASCT2, filled arrowheads indicate neurons expressing SERT only and empty arrowheads indicate neurons expressing ASCT2 only. Scale bar, 100 μm. (B and C) Co-immunoprecipitation of native SERT with native ASCT2 expressed in mice brain. Inputs (left-hand panels) represent 1% of the protein amount used for immunoprecipitation. Immunoprecipitations were performed with either the anti-SERT antibody (B) or the anti-mouse ASCT2 antibody (C). Non-immune purified rabbit IgG was used as control. Representative results of three independent experiments are illustrated. Mw, molecular mass (indicated in kDa). IP, immunoprecipitation.

Inhibition of SERT plasma membrane localization and 5-HT uptake by ASCT2 expression

Co-expression of FLAG-tagged ASCT2 with YFP-tagged SERT in HEK-293 cells yielded a marked increase in overall ASCT2 level, as assessed by Western blotting using an antibody recognizing human ASCT2 (Supplementary Figure S2). ASCT2 overexpression strongly modified the pattern of SERT immunoreactive signals detected by Western blotting: although under both experimental conditions SERT appears as two major immunoreactive bands of molecular masses of 80 and 115 kDa respectively, the upper band was predominant when SERT was expressed alone, whereas the lower-molecular-mass form was mainly detected in cells co-expressing SERT and ASCT2 (Figure 1C). Moreover, the treatment of solubilized protein extracts from HEK-293 cells expressing SERT alone or co-expressing SERT and ASCT2 with PNGase F (peptide N-glycosidase F) induced a shift in SERT immunoreactivity towards a lower molecular mass (∼70 kDa) and no more difference in the pattern of immunoreactive signals was detected between both PNGase F-treated samples (Figure 1D). Collectively, these results indicate that the predominant forms of SERT in HEK-293 cells are N-glycosylated and are consistent with the glycosylation pattern of SERT observed previously [8]. Moreover, they show that ASCT2 expression causes a marked modification in the proportion of glycosylated forms of SERT, thus suggesting that ASCT2 affects SERT maturation.

In HEK-293 cells co-expressing SERT and ASCT2, the maximal velocity of 5-HT uptake was decreased (−56.5±10.4%, n=8, P<0.01), compared with cells expressing SERT alone (Figure 3A). In line with these findings, biotinylation experiments showed that the amount of SERT at the cell surface (biotinylated) was decreased (−59.7±6.5%, n=3, P<0.05) in cells co-expressing SERT and ASCT2, compared with cells expressing SERT alone (Figure 3B). Correspondingly, an increase in intracellular SERT (non-biotinylated) was found in cells co-expressing SERT and ASCT2. Immunocytochemistry experiments combined with Apotome fluorescence microscopy confirmed a change in SERT cellular distribution upon ASCT2 overexpression: an important fraction of SERT was detected in intracellular compartments, where it was co-localized with ASCT2, in cells overexpressing ASCT2, whereas it was mainly located at the plasma membrane in cells expressing endogenous levels of ASCT2 (Figure 4). Furthermore, ASCT2 expression specifically affected plasma membrane localization of fully glycosylated SERT, whereas the subcellular distribution of partially glycosylated/non-glycosylated forms was not significantly affected (Figure 3B). Collectively, these observations suggest that ASCT2 decreases 5-HT uptake activity by inhibiting its maturation and, consequently, its export to the plasma membrane. Co-expression of ASCT2 also induced a significant increase in the apparent affinity of SERT for 5-HT transport (Km=2.8±0.7 μM in cells co-expressing SERT and ASCT2 compared with 5.8±0.8 μM for cells transfected with SERT alone, P<0.001). This result is consistent with previous findings indicating that the apparent affinity of the transporter for substrate and inhibitors is decreased in cells with high levels of SERT expression at the plasma membrane and suggesting an interaction of SERT with endogenous factors that modulate its transport activity and alter ligand potency [58,59].

ASCT2 expression inhibits SERT plasma membrane localization and 5-HT transport activity

Figure 3
ASCT2 expression inhibits SERT plasma membrane localization and 5-HT transport activity

(A) [3H]5-HT uptake in HEK-293 cells expressing SERT alone or co-expressing SERT and ASCT2. Representative concentration-dependent curves are shown. Vmax and Km of 5-HT uptake, expressed as a percentage of the values measured in cells transfected with SERT alone (control, 8.73±1.95 pmol/min per 105 cells and 5.84±0.83 μM respectively), are means±S.E.M. for three independent experiments. **P<0.01, ***P<0.001 compared with cells expressing SERT alone. (B) Cell-surface biotinylation assay performed on HEK-293 cells expressing SERT in the absence or presence of ASCT2. Total, non-biotinylated and biotinylated SERT were detected by Western blotting using monoclonal anti-GFP antibody. Blots representative of three independent experiments are shown. The histograms represent biotinylated SERT normalized to the amount of total SERT, and the proportion of fully glycosylated SERT and partially glycosylated/non-glycosylated SERT in the biotinylated fraction respectively. Results are means±S.E.M. for densitometric analyses of blots obtained in three independent experiments performed on different sets of cultured cells. *P<0.05, ***P<0.001 compared with cells expressing SERT alone. Mw, molecular mass (indicated in kDa).

Figure 3
ASCT2 expression inhibits SERT plasma membrane localization and 5-HT transport activity

(A) [3H]5-HT uptake in HEK-293 cells expressing SERT alone or co-expressing SERT and ASCT2. Representative concentration-dependent curves are shown. Vmax and Km of 5-HT uptake, expressed as a percentage of the values measured in cells transfected with SERT alone (control, 8.73±1.95 pmol/min per 105 cells and 5.84±0.83 μM respectively), are means±S.E.M. for three independent experiments. **P<0.01, ***P<0.001 compared with cells expressing SERT alone. (B) Cell-surface biotinylation assay performed on HEK-293 cells expressing SERT in the absence or presence of ASCT2. Total, non-biotinylated and biotinylated SERT were detected by Western blotting using monoclonal anti-GFP antibody. Blots representative of three independent experiments are shown. The histograms represent biotinylated SERT normalized to the amount of total SERT, and the proportion of fully glycosylated SERT and partially glycosylated/non-glycosylated SERT in the biotinylated fraction respectively. Results are means±S.E.M. for densitometric analyses of blots obtained in three independent experiments performed on different sets of cultured cells. *P<0.05, ***P<0.001 compared with cells expressing SERT alone. Mw, molecular mass (indicated in kDa).

ASCT2 expression but not ASCT2 activity changes plasma membrane localization of SERT in HEK-293 cells

Figure 4
ASCT2 expression but not ASCT2 activity changes plasma membrane localization of SERT in HEK-293 cells

HEK-293 cells were transfected with plasmids encoding YFP-tagged SERT (green) or FLAG-tagged ASCT2 (red) alone or in combination. Cells were treated with vehicle or 2 mM D-threonine (D-Thr), either for 20 min or 48 h (in that case, D-threonine was added to the culture medium immediately after transfection). For cells co-expressing SERT and ASCT2, merged images are shown in the bottom three rows. Representative cells from three independent cultures are illustrated. Scale bar, 20 μm.

Figure 4
ASCT2 expression but not ASCT2 activity changes plasma membrane localization of SERT in HEK-293 cells

HEK-293 cells were transfected with plasmids encoding YFP-tagged SERT (green) or FLAG-tagged ASCT2 (red) alone or in combination. Cells were treated with vehicle or 2 mM D-threonine (D-Thr), either for 20 min or 48 h (in that case, D-threonine was added to the culture medium immediately after transfection). For cells co-expressing SERT and ASCT2, merged images are shown in the bottom three rows. Representative cells from three independent cultures are illustrated. Scale bar, 20 μm.

To determine whether endogenously expressed ASCT2 modulates SERT transport activity, we silenced expression of ASCT2 in HEK-293 cells using an siRNA (Figure 5A). Depletion of ASCT2 increased the maximal 5-HT uptake and decreased the apparent affinity of SERT for 5-HT transport (Vmax=6.40±0.78 pmol/min per 105 cells and Km=3.18±0.19 μM in cells transfected with the ASCT2 siRNA compared with Vmax=3.98±0.45 pmol/min per 105 cells and Km=2.59±0.18 μM in cells transfected with control siRNA, n=4, P<0.01 and P<0.05 respectively; Figure 5B). We also found an overall increase in SERT expression in ASCT2 siRNA-transfected cells, compared with cells transfected with control siRNA (+60.6±8.6%, n=4, P<0.01; Figure 5A), with a most prominent increase in the higher-molecular-mass band corresponding to the mature form of SERT (+70.2%, P<0.01; Figure 5A), whereas there was no significant difference in the lower-molecular-mass band corresponding to the partially glycosylated/non-glycosylated form (P=0.154; Figure 5A). These results corroborate those obtained in ASCT2-overexpression experiments and suggest that an increase in SERT expression and/or plasma membrane localization might contribute to the enhanced 5-HT uptake in cells depleted of ASCT2.

Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in HEK-293 cells

Figure 5
Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in HEK-293 cells

(A) HEK-293 cells were co-transfected with the plasmid encoding SERT and either control or ASCT2 siRNAs. Total protein extracts were analysed by Western blotting using polyclonal anti-ASCT2 antibody, monoclonal anti-GFP antibody and polyclonal anti-GAPDH antibody. The histogram represents the amount of total SERT, fully glycosylated SERT and partially glycosylated/non-glycosylated SERT. Results are means±S.E.M. for densitometric analyses of blots obtained in three independent experiments performed on different sets of cultured cells. *P<0.05, **P<0.01 compared with cells expressing SERT alone. (B) [3H]5-HT uptake in HEK-293 cells expressing SERT in the presence of control or ASCT2 siRNA. A representative experiment is shown. Vmax and Km of 5-HT uptake, expressed as a percentage of the value measured in cells transfected with control siRNA (3.98±0.45 pmol/min per 105 cells and 2.59±0.18 μM respectively), are means±S.E.M. for three independent experiments. *P<0.05, **P<0.01 compared with cells transfected with control siRNA. (C) Vmax of [3H]5-HT uptake in HEK-293 cells transfected with the plasmid encoding SERT and either control or ASCT2 siRNA, and treated or not with 2 mM D-threonine (D-Thr). Results, expressed as percentages of the values measured in control siRNA-transfected cells in the absence of D-threonine (Vehicle), are means±S.E.M. for three independent experiments. **P<0.01 compared with cells transfected with control siRNA and treated with vehicle. n.s., not significant. (D) HEK-293 cells expressing YFP-tagged SERT alone or co-expressing YFP-tagged SERT and FLAG-tagged ASCT2 were incubated for 20 min or 48 h in the absence or presence of 2 mM D-threonine. Total protein extracts were analysed by Western blotting using monoclonal anti-GFP antibody. A representative Western blot of three independent experiments performed on different cultures is shown. Mw, molecular mass (indicated in kDa).

Figure 5
Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in HEK-293 cells

(A) HEK-293 cells were co-transfected with the plasmid encoding SERT and either control or ASCT2 siRNAs. Total protein extracts were analysed by Western blotting using polyclonal anti-ASCT2 antibody, monoclonal anti-GFP antibody and polyclonal anti-GAPDH antibody. The histogram represents the amount of total SERT, fully glycosylated SERT and partially glycosylated/non-glycosylated SERT. Results are means±S.E.M. for densitometric analyses of blots obtained in three independent experiments performed on different sets of cultured cells. *P<0.05, **P<0.01 compared with cells expressing SERT alone. (B) [3H]5-HT uptake in HEK-293 cells expressing SERT in the presence of control or ASCT2 siRNA. A representative experiment is shown. Vmax and Km of 5-HT uptake, expressed as a percentage of the value measured in cells transfected with control siRNA (3.98±0.45 pmol/min per 105 cells and 2.59±0.18 μM respectively), are means±S.E.M. for three independent experiments. *P<0.05, **P<0.01 compared with cells transfected with control siRNA. (C) Vmax of [3H]5-HT uptake in HEK-293 cells transfected with the plasmid encoding SERT and either control or ASCT2 siRNA, and treated or not with 2 mM D-threonine (D-Thr). Results, expressed as percentages of the values measured in control siRNA-transfected cells in the absence of D-threonine (Vehicle), are means±S.E.M. for three independent experiments. **P<0.01 compared with cells transfected with control siRNA and treated with vehicle. n.s., not significant. (D) HEK-293 cells expressing YFP-tagged SERT alone or co-expressing YFP-tagged SERT and FLAG-tagged ASCT2 were incubated for 20 min or 48 h in the absence or presence of 2 mM D-threonine. Total protein extracts were analysed by Western blotting using monoclonal anti-GFP antibody. A representative Western blot of three independent experiments performed on different cultures is shown. Mw, molecular mass (indicated in kDa).

To explore further whether the expression of ASCT2 likewise affects activity of native SERT expressed in neurons, we silenced ASCT2 expression in mesencephalon neurons from mice embryos in primary culture. In these cultures, SERT is expressed in a minority of neurons (∼2%) and 5-HT uptake can be blocked by a low concentration of fluoxetine, indicating SERT-operated 5-HT transport [31]. We infected cultured neurons with a lentivirus expressing an ASCT2 shRNA to ensure a more efficient ASCT2 down-regulation than that expected by transfecting neurons with an siRNA. The ability of the shRNA to down-regulate ASCT2 expression in primary neurons was verified by qRT-PCR. These experiments showed a strong down-regulation of ASCT2 mRNA in ASCT2 shRNA-infected neurons (−94.29±1.18%, n=3, P<0.001; Figure 6A), compared with neurons infected with a lentivirus expressing a control shRNA. Reminiscent of the results obtained in HEK-293 cells, the velocity of 5-HT uptake was also increased by down-regulating expression of ASCT2 in neurons (+58.60±0.81% compared with control shRNA-expressing neurons, P<0.05; Figure 6B). Collectively, our results indicate that ASCT2 expression affects maturation and cell-surface localization of SERT in neurons with a concomitant reduction in its 5-HT uptake activity. Further studies will be necessary to establish whether the modulation of SERT transport activity is mediated by physically associated ASCT2, an issue requiring the identification of residues or sequences in both partner proteins that are critical for their interaction.

Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in mesencephalon neurons

Figure 6
Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in mesencephalon neurons

(A) Analysis of ASCT2 mRNA levels by qRT-PCR in primary mesencephalon neurons infected with a lentivirus expressing control or ASCT2 shRNA. Results are means±S.E.M. of triplicate determinations. (B) [3H]5-HT uptake in mouse mesencephalon neurons infected with a lentivirus expressing control or ASCT2 shRNA. Results are means±S.E.M. from a representative experiment. Two other experiments yielded similar results. **P<0.01 compared with control shRNA-infected neurons. (C and D) Effects of increasing concentrations of D-threonine (D-Thr) on [3H]alanine (C) or [3H]glutamine (D) uptake (final concentration 20 nM) in neurons. (E) Effect of D-threonine on [3H]5-HT uptake in neurons. Results, expressed as percentage values measured in the absence of D-threonine (0.33±0.06 pmol/min per 106 cells), are means±S.E.M. for five independent experiments performed on different sets of cultured neurons. *P<0.05, ***P<0.001 compared with 5-HT uptake in the absence of D-threonine.

Figure 6
Silencing ASCT2 expression and ASCT2 activity inhibition decrease 5-HT uptake via SERT in mesencephalon neurons

(A) Analysis of ASCT2 mRNA levels by qRT-PCR in primary mesencephalon neurons infected with a lentivirus expressing control or ASCT2 shRNA. Results are means±S.E.M. of triplicate determinations. (B) [3H]5-HT uptake in mouse mesencephalon neurons infected with a lentivirus expressing control or ASCT2 shRNA. Results are means±S.E.M. from a representative experiment. Two other experiments yielded similar results. **P<0.01 compared with control shRNA-infected neurons. (C and D) Effects of increasing concentrations of D-threonine (D-Thr) on [3H]alanine (C) or [3H]glutamine (D) uptake (final concentration 20 nM) in neurons. (E) Effect of D-threonine on [3H]5-HT uptake in neurons. Results, expressed as percentage values measured in the absence of D-threonine (0.33±0.06 pmol/min per 106 cells), are means±S.E.M. for five independent experiments performed on different sets of cultured neurons. *P<0.05, ***P<0.001 compared with 5-HT uptake in the absence of D-threonine.

Control of 5-HT uptake via SERT by ASCT2 transport activity

To explore whether ASCT2 transport activity regulates 5-HT uptake via SERT, we treated cells with D-threonine, an ASCT2 inhibitor [60]. Consistent with previous findings, D-threonine concentration-dependently inhibited [3H]alanine and [3H]glutamine uptake in both HEK-293 cells (pIC50=1.98±0.03 and 1.91±0.11 respectively, n=3; Supplementary Figure S3) and primary mesencephalon neurons (pIC50=3.51±0.09 and 3.24±0.41 respectively; Figures 6C and 6D). Acute treatment of HEK-293 cells with D-threonine (2 mM for 20 min) significantly increased maximal 5-HT uptake (+28.60±7.47%, n=3, P<0.01; Figure 5C), when compared with vehicle-treated cells. D-Threonine did not increase further the augmented velocity of 5-HT uptake observed in ASCT2 siRNA-treated cells (Figure 5C), strongly suggesting that the D-threonine-elicited modulation of SERT activity is mediated by the blockade of ASCT2. In addition, neither acute (20 min) nor prolonged (48 h) D-threonine treatment altered the glycosylation pattern (Figure 5D) and plasma membrane localization of SERT (Figure 4), suggesting that neutral amino acid transport activity of ASCT2 does not affect SERT maturation. Consistent with the results obtained in transfected HEK-293 cells, exposure of primary mesencephalon neurons to D-threonine (0.5 or 2 mM for 20 min) enhanced 5-HT uptake into neurons, compared with vehicle-treated neurons (Figure 6E). This suggests that ASCT2 activity modulates 5-HT transport activity of recombinant SERT expressed in HEK-293 cells and native SERT expressed in 5-HT neurons.

Collectively, the present findings suggest that ASCT2 expression and activity might affect SERT's functional status through two distinct mechanisms. (i) ASCT2 expression impairs SERT maturation, a process leading to SERT sequestration in intracellular compartments and, accordingly, to a decrease in the availability of functional SERT and 5-HT uptake. This effect is independent of ASCT2 functional activity, as SERT plasma membrane insertion was not affected by treatment of cells with the ASCT2 inhibitor D-threonine, and probably results from physical association of ASCT2 with SERT within intracellular compartments. (ii) ASCT2 activity affects SERT 5-HT transport activity independently of an effect upon SERT maturation/insertion, suggesting functional interactions between SERT and ASCT2 with heteromers present at the plasma membrane, which depend on the presence of substrates or inhibitors of each transporter protein. The impact of ASCT2 expression and activity upon 5-HT transmission would thus be of considerable interest to explore in future studies. Comparing cerebral 5-HT uptake in wild-type and ASCT2-knockout mice [61] and in mice exposed or not to ASCT2 modulators might certainly help to resolve this important issue.

Differential modulation of monoamine transporters by ASCT2

To explore whether ASCT2 interacts with and modulates activity of other monoamine transporters, we co-expressed YFP-tagged versions of SERT, DAT or NET with FLAG-tagged ASCT2 in HEK-293 cells and first compared their ability to co-immunoprecipitate with ASCT2. Although the three transporters co-immunoprecipitated with ASCT2, the amounts of DAT and NET in ASCT2 immunoprecipitates were much lower compared with the amount of co-immunoprecipitated SERT (Figure 7A). This result suggests that DAT and NET interact more weakly than SERT with ASCT2 or that the stoichiometry of their association with ASCT2 is weaker, when compared with that of SERT interaction with ASCT2. Moreover, contrasting with its inhibitory influence upon SERT activity, co-expression of ASCT2 slightly but significantly increased dopamine uptake (+19.69±3.64%, n=3, P<0.05) without affecting its apparent affinity for DAT (P=0.292; Figure 7B), whereas neither the uptake of noradrenaline nor its apparent affinity for NET were significantly affected (P=0.814 and 0.210 respectively; Figure 7C). Collectively, these findings show that, among the three monoamine transporters, ASCT2 preferentially interacts with and specifically inhibits the activity of SERT. This suggests that ASCT2 might preferentially control physiological functions mediated by the serotonergic system, compared with the dopaminergic and noradrenergic systems. Nevertheless, monoaminergic systems are strongly interconnected and their projections to the forebrain create an intricate network of overlapping innervations. This permits complex functional interactions that underlie the modulation of key brain functions, such as executive function, cognition, emotions and mood [62]. Consequently, ASCT2 might also indirectly affect the activity and some physiological functions of dopaminergic and noradrenergic systems in spite of its modest effects on the transport activities of DAT and NET.

ASCT2 differentially modulates activity of monoamine transporters

Figure 7
ASCT2 differentially modulates activity of monoamine transporters

(A) HEK-293 cells were transfected with plasmids encoding GFP, YFP-tagged SERT, YFP-tagged DAT or YFP-tagged NET, alone or in combination with the plasmid encoding FLAG-tagged ASCT2. Proteins were immunoprecipitated with monoclonal anti-FLAG antibody conjugated to Sepharose beads. Inputs and immunoprecipitates were analysed by Western blotting using monoclonal anti-GFP and polyclonal anti-FLAG antibodies to detect monoamine transporters and ASCT2 respectively. Inputs represent 2% of the total protein amount used for immunoprecipitations. Equal loading in inputs was assessed by Western blotting using anti-GFAP (glial fibrillary acid protein) antibody. Blots are representative of three independent experiments. Mw, molecular mass (indicated in kDa) (B) [3H]Dopamine uptake in HEK-293 cells expressing DAT alone or co-expressing DAT and ASCT2. (C) [3H]Noradrenaline uptake in HEK-293 cells co-expressing NET and ASCT2. Vmax and Km of dopamine and noradrenaline uptake, expressed as percentage values in cells expressing DAT alone (4.67±1.49 pmol/min per 105 cells and 8.03±2.00 μM) or NET alone (1.69±0.65 pmol/min per 105 cells and 3.92±0.66 μM) respectively, are means±S.E.M. for three independent experiments. *P<0.05 compared with cells expressing DAT alone.

Figure 7
ASCT2 differentially modulates activity of monoamine transporters

(A) HEK-293 cells were transfected with plasmids encoding GFP, YFP-tagged SERT, YFP-tagged DAT or YFP-tagged NET, alone or in combination with the plasmid encoding FLAG-tagged ASCT2. Proteins were immunoprecipitated with monoclonal anti-FLAG antibody conjugated to Sepharose beads. Inputs and immunoprecipitates were analysed by Western blotting using monoclonal anti-GFP and polyclonal anti-FLAG antibodies to detect monoamine transporters and ASCT2 respectively. Inputs represent 2% of the total protein amount used for immunoprecipitations. Equal loading in inputs was assessed by Western blotting using anti-GFAP (glial fibrillary acid protein) antibody. Blots are representative of three independent experiments. Mw, molecular mass (indicated in kDa) (B) [3H]Dopamine uptake in HEK-293 cells expressing DAT alone or co-expressing DAT and ASCT2. (C) [3H]Noradrenaline uptake in HEK-293 cells co-expressing NET and ASCT2. Vmax and Km of dopamine and noradrenaline uptake, expressed as percentage values in cells expressing DAT alone (4.67±1.49 pmol/min per 105 cells and 8.03±2.00 μM) or NET alone (1.69±0.65 pmol/min per 105 cells and 3.92±0.66 μM) respectively, are means±S.E.M. for three independent experiments. *P<0.05 compared with cells expressing DAT alone.

In conclusion, the observations of the present study suggest that SERT oligomers are part of larger complexes of transmembrane proteins, which include transporters of different solute carrier families, such as ASCT2. They also identify ASCT2 as an important regulator of SERT subcellular distribution and 5-HT transport activity, thus suggesting that reuptake of 5-HT might be modulated by the extracellular concentrations of small neutral amino acids. Several lines of evidence indicate an enhanced expression and activity of ASCT2 in various cancers, especially in those derived from tissues that possess a strong glutamine-dependent metabolic profile such as the brain, where intercellular glutamine cycles function as part of their normal physiological role [63]. They provided the impetus for the development of new ASCT2 inhibitors for cancer therapy. In the light of the observations of the present study, which show that down-regulating ASCT2 expression or inhibiting its activity increases 5-HT transport activity of SERT, the development of those inhibitors should take into consideration possible side effects linked to the disruption of 5-HT transmission following prolonged use.

AUTHOR CONTRIBUTION

Pascal Seyer designed and performed experiments, analysed the data and wrote the paper. Franck Vandermoere performed the MS analyses and analysed the data. Elisabeth Cassier performed the co-immunoprecipitation and immunohistochemistry experiments. Joël Bockaert supported the project and wrote the paper. Philippe Marin supported the project, designed the experiments, analysed the data and wrote the paper.

MS analyses were carried out using the MS facilities of the Functional Proteomics Platform of Montpellier Languedoc-Roussillon (http://www.fpp.cnrs.fr/). Monoamine and amino acid uptake studies were carried out using the facilities of the Pharmacology-Screening-Interactome Arpege platform (http://www.arpege.cnrs.fr/). We also thank Anne Le Digarcher (Montpellier Genomix platform, http://www.mgx.cnrs.fr/) for technical help in qRT-PCR experiments.

FUNDING

This work was supported by grants from the Fondation pour la Recherche Médicale (Contracts Equipe FRM 2005 and 2009), Agence Nationale de la Recherche [grant number ANR-08-MNPS-034], Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de Montpellier and la Région Languedoc-Roussillon.

Abbreviations

     
  • AP

    affinity purification

  •  
  • ASCT2

    alanine–serine–cysteine–threonine 2

  •  
  • DAT

    dopamine transporter

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • ER

    endoplasmic reticulum

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HEK

    human embryonic kidney

  •  
  • HSP

    heat-shock protein

  •  
  • 5-HT

    5-hydroxytryptamine

  •  
  • iBAQ

    intensity-based absolute quantification

  •  
  • MacMARCKS

    macrophage myristoylated alanine-rich C-kinase substrate

  •  
  • NA

    numerical aperture

  •  
  • NET

    noradrenaline transporter

  •  
  • PGRMC1

    membrane-associated progesterone receptor component 1

  •  
  • PKC

    protein kinase C

  •  
  • PNGase F

    peptide N-glycosidase F

  •  
  • PP2A

    protein phosphatase 2A

  •  
  • PP2Ac

    catalytic subunit of PP2A

  •  
  • qRT-PCR

    quantitative reverse transcription–PCR

  •  
  • SCAMP

    secretory carrier membrane protein

  •  
  • SERT

    serotonin transporter

  •  
  • SLC

    solute carrier

  •  
  • TM

    transmembrane region

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Supplementary data