title: Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter.
abstract: Na+/Cl--dependent neurotransmitter transporters form constitutive oligomers. The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer. To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus. Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET). Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity. In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini. Hence, a separate interaction domain (i.e. distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT. This was confirmed by expressing the amino- and carboxyl-terminal halves of hSERT. These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET. Hence, both the carboxyl and amino termini contain elements that drive oligomerization. By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and beta-lactamase protein fragment complementation assay. TM1/2 was also found to self-associate. Thus, oligomerization of hSERT involves at least two discontinuous interfaces. The currently identified interaction sites drive homophilic interactions. This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers.
authors.affiliation: Institute of Pharmacology, University of Vienna Medical School, Währinger Strasse 13A, A-1090 Vienna, Austria.
authors.forename: Herwig
authors.initials: H
authors.lastname: Just
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Schmid
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D012701:Serotonin / Q000737:chemistry; D050486:Serotonin Plasma Membrane Transport Proteins; D013997:Time Factors; D014162:Transfection; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; D012701:Serotonin; D004247:DNA; D001618:beta-Lactamases