title: The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation.
abstract: The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the "long loop" recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.
authors: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, and University of Copenhagen, DK-2200 Copenhagen, Denmark; Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors: Troels
authors: T
authors: Rahbek-Clemmensen
authors: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, and University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors: Tina
authors: T
authors: Bay
authors: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, and University of Copenhagen, DK-2200 Copenhagen, Denmark; Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors: Jacob
authors: J
authors: Eriksen
authors: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, and University of Copenhagen, DK-2200 Copenhagen, Denmark; Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address: gether@sund.ku.dk.
authors: Ulrik
authors: U
authors: Gether
authors: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, and University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors: Trine Nygaard
authors: TN
authors: Jørgensen
mesh_terms: D002273:Carcinogens / Q000494:pharmacology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D059748:Proteolysis; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
chemical_list: D002273:Carcinogens; D050486:Serotonin Plasma Membrane Transport Proteins; D013755:Tetradecanoylphorbol Acetate; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C493230:SLC6A4 protein, human; C000716463:rab7 GTP-binding proteins, human
keywords: Cellular Regulation; Endocytosis; Intracellular Trafficking; Monoamine Transporter; Protein Kinase C (PKC); Protein Sorting; Serotonin Transporter