title: Mutations in the carboxyl-terminal SEC24 binding motif of the serotonin transporter impair folding of the transporter.
abstract: The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.
authors.affiliation: Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Währinger Str. 13a, A-1090 Vienna, Austria.
authors.forename: Ali
authors.initials: A
authors.lastname: El-Kasaby
authors.forename: Herwig
authors.initials: H
authors.lastname: Just
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Malle
authors.forename: Peggy C
authors.initials: PC
authors.lastname: Stolt-Bergner
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007099:Imipramine / Q000737:chemistry; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry; D009154:Mutation; D018377:Neurotransmitter Agents / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012701:Serotonin / Q000737:chemistry; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry*; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D018832:Molecular Chaperones; D018377:Neurotransmitter Agents; C530641:SEC24D protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D033921:Vesicular Transport Proteins; D012701:Serotonin; D007099:Imipramine