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. 2011 Apr;14(4):469-77.
doi: 10.1038/nn.2781. Epub 2011 Mar 13.

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

M Laura Cremona  1 , Heinrich J G Matthies, Kelvin Pau, Erica Bowton, Nicole Speed, Brandon J Lute, Monique Anderson, Namita Sen, Sabrina D Robertson, Roxanne A Vaughan, James E Rothman, Aurelio Galli, Jonathan A Javitch, Ai Yamamoto
Affiliations

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

M Laura Cremona et al. Nat Neurosci. 2011 Apr.

Erratum in

  • Nat Neurosci. 2011 Dec;14(2):1617

Abstract

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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Figures

Fig.1
Fig.1. PKC triggers endocytosis of heterologously and endogenously expressed DAT
a,b. EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.1 µM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa-Fluor 633 labeled secondary antibodies were used to prevent overlap with YFP. Scale bar = 10 µm. c. Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 µM PMA (+PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope, Chemicon), and Alexa-Fluor 568 secondary. PMA led to significant internalization of DAT (‘Internalization Index,’ as described in Methods, Ctrl: 0.076±0.013; vs. PMA: 0.309±0.052). (One-way ANOVA; p < 0.001). Scale bar = 20 µm. d. Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 µM PMA or Ctrl for 1 hr, then cell surface biotinylated to determine DAT surface levels. ‘DAT expression levels (% Surface)’ indicates the relative amount of DAT at the cell surface, and was calculated as the ratio between the integrated densities of the surface levels of DAT after treatment (corrected for total levels of DAT) to the surface levels of DAT before treatment (corrected for total levels of DAT). Bars represent Mean + St. Dev. PMA treatment significantly decreased DAT surface levels (p = 0.0330). Complete blots can be found in Figure S9.
Fig.2
Fig.2. Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosis
Flot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.
Fig.3
Fig.3. Flot1 is required for the PKC-triggered endocytosis of EM4-YFP-DAT and Hela-eGFP-EAAT2
a–b. Flot1 is required for PKC-triggered endocytosis of DAT. EM4-YFP-DAT cells transfected with siRNA against Flot1 (siFlot1) failed to internalize DAT despite exposure to 1 µM PMA for 30 min. a. Confocal microscopy (n = 4). b. Cell surface biotinylation (n = 4). Data are plotted as described (Fig.1d). PMA treatment did not lead to a significant decrease of cell surface DAT after Flot1 depletion (p = 0.226). c–d. Flot1 is required for PKC-triggered endocytosis of EAAT2. c. GFP-EAAT2 internalized to EAA1-positive structures after 0.5 µM PMA for 30 min. (n = 3). d. Cell surface biotinylation (n = 3). PMA treatment did not lead to a significant decrease of cell surface EAAT2 after Flot1 depletion (p = 0.104). Cells were treated with 0.5 µM PMA for 30 min. Complete statistics can be found in Supporting Materials. e. Confocal microscopy revealed that DAT (green) internalized into Flot1-positive vesicles (red) after exposure to 1 µM PMA for 30 min (overlay in yellow). Scale bars = 10 µm. Complete blots can be found in Figure S11.
Fig.4
Fig.4. Flot1 must be palmitoylated on residue Cys34
a. Schematic representation of Flot1. The conserved cysteine Cys34 is found within the first hydrophobic domain in the PHB homology domain. Four predicted PKC-phosphorylation sites are also noted. b. Overexpression of Flot1(C34A) in EM4-YFP-DAT inhibited PKC-triggered internalization of DAT (n = 6 experiments). Cells transiently transfected with Flot1-mRFP, Flot1(C34A)-mRFP, or mRFP alone (Ctrl), were exposed to 1 µM PMA for 30 min. Representative images shown. Scale bar = 10 µm. c. Cell surface biotinylation of DAT after PMA treatment in the presence or absence of Gö6850. C34A inhibited PMA-triggered internalization (left panels, −Gö6850), and also failed to diminish Gö6850 inhibition (right panels, +Gö6850). d. Quantification of DAT surface expression of c., calculated as described (Fig.1d) (n = 3). e. Palmitoylation is not required for multimerization of Flot1. EM4-YFP-DAT cells transiently transfected with mRFP, mRFP-Flot1 or mRFP-Flot1(C34A). Flot1 fusion proteins were immunoprecipitated with an antibody against mRFP, and probed for endogenous Flot1 (left panel). Flot1(C34A)-mRFP still co-IPed with endogenous Flot1 (n = 3). mRFP alone did not co-IP with Flot1 (right panel). f. Palmitoylation is required for proper complex formation of Flot1 and DAT. Flot1(C34A) pulled-down significantly less DAT than non-mutagenized Flot1. 1 µM PMA for 30 min significantly increased the amount of DAT that co-IPs with WT Flot1 (p = 0.0148). This effect was also abrogated by the C34A mutation (p = 0.1022) (n = 4). Complete statistics can be found in Supporting Materials. Complete blots can be found in Figure S12.
Fig.5
Fig.5. Flot1 is required for the membrane raft localization of DAT but not for transport of DA
Sucrose gradients revealed that Flot1 was required to maintain DAT in membrane rafts. EM4-YFP-DAT cells were lysed with 1% w/v Brij58 and fractionated across a discontinuous sucrose gradient. Ten equivolume fractions were collected from top (1) to bottom (10) of the gradient. Four different conditions were run: a. Control conditions (Flot1); b. Flot1(C34A) transfection; c. siFlot1 transfection; and d. nystatin (25 µg/mL) (Nys). (n = 3).e. A graphic representation of integrated densities across fractions for each condition is shown for DAT and TfR. f. The transport of DA by DAT does not require the presence of Flot1. EM4-YFP-DAT cells were transfected as indicated, exposed to vehicle, then DA uptake assays were performed as described in Materials. Data is represented as ‘[3H]-DA uptake (%WT)’. Neither the palmitoylation-deficient mutant nor depletion of Flot1 significantly altered DA uptake (n = 4, p = 0.5770). Please also refer to Table 1. Complete blots can be found in Figure S13.
Fig.6
Fig.6. PKC-triggered internalization of DAT requires phosphorylation on Flot1Ser315
a. PMA (1µM, 30min) promotes association of Flot1 with DAT. Bars represent the Mean ratio between integrated densities of endogenous Flot1 to Flot1-mRFP+SEM (n=4). PMA significantly increased mRFP-Flot1 and Flot1 to co-IP with DAT (ANOVA, p = 0.0421). Cav1 did not immunoprecipitate with this complex. b. Metabolic labeling reveals that Flot1 was phosphorylated in response to PMA. EM4-YFP-DAT were treated with vehicle (Ctrl), 1µM PMA + vehicle (+PMA) or 1µM PMA + 100nM Gö6850 pre-incubation (+Go) (n = 2). Bars represent the integrated density of 32P incorporation normalized to Flot1 levels+SEM. ANOVA reveals +PMA led to a significant increase in 32P incorporation (p = 0.0474) that was inhibited by Gö6850 (p = 0.0017). S315A mutation inhibited this PMA-triggered increase (p = 0.1610). Overall 32P incorporation was also significantly less in S315A mutant (p < 0.001). c.–e.. PMA (1µM, 30min)-triggered internalization of DAT requires Flot1Ser315. Flot1(S315A) overexpression inhibited DAT internalization while Flot1(S54A) did not. c. DAT is internalized in non-transfected (white arrows) and Flot1(S54A) transfected cells (yellow arrows), but not in Flot1(S315) transfected cells (yellow arrow). ‘% Cells with internalization’ was determined as described (Fig.2a) (200 cells/condition) (ANOVA, p < 0.01). Scale bar = 5µm. d. Surface protein biotinylation and quantification of DAT (n = 3) (ANOVA, p < 0.01). e. Alignment of region surrounding Flot1Ser315. Ser315 is largely conserved across species (*, bold). Complete statistics can be found in Supporting Materials. Complete blots can be found in Figure S14.
Fig.7
Fig.7. Flot1 is required for PKC-triggered internalization and AMPH-induced reverse transport of DA in primary DAergic neurons
a. Representative DAT-mediated transient currents recorded from DA neurons transduced with lentiviri carrying shCtrl or shFlot1 following a voltage jump to −140 mV from a holding potential of −20 mV. The integral of the DAT-mediated transient current is proportional to the number of transporters on the cell surface. Transient currents were obtained first with vehicle, then in the presence of PMA. Bars represent the mean ratios of PMA transient currents to pre-PMA responses in the same cell. PMA did not reduce DAT-mediated transient currents in shFlot1 neurons, suggesting that Flot1 is necessary for PMA-triggered DAT trafficking. b. Loss of Flot1 leads to an inhibition of AMPH-mediated DA efflux. Neurons were loaded via the patch pipette with 2 mM DA and 30 mM Na+. Amperometric current-voltage relationship was obtained by stepping the voltage in 20 mV intervals from 0 mV to +100 mV from a holding potential of −60 mV. DAT-mediated DA efflux is defined as the current recorded in the presence of AMPH, minus the current recorded after the addition of cocaine to the bath with AMPH still present from shFlot1 (red, n = 4) and shCtrl (black, n = 4). Values are reported as mean ± SEM (* indicates p < 0.05). shFlot1 significantly reduced DA efflux as compared to shCtrl at voltages greater than +40 mV (* p < 0.05, ** p < 0.01, One-way ANOVA followed by Bonferroni posthoc tests).
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