title: A cytosolic relay of heat shock proteins HSP70-1A and HSP90β monitors the folding trajectory of the serotonin transporter.
abstract: Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and the Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, 35516 Mansoura, Egypt.
authors.forename: Ali
authors.initials: A
authors.lastname: El-Kasaby
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors.forename: Florian
authors.initials: F
authors.lastname: Koban
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and sonja.sucic@meduniwien.ac.at.
authors.forename: Sonja
authors.initials: S
authors.lastname: Sucic
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007050:Ibogaine / Q000031:analogs & derivatives / Q000737:chemistry; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; C483693:Hspa14 protein, human; D034741:RNA, Small Interfering; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D007050:Ibogaine; C094385:noribogaine; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases
keywords: Glycosylation; Heat Shock Protein (HSP); Heat Shock Protein 90 (Hsp90); Serotonin; Serotonin Transporter; Trafficking; Transporter