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A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter

The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4)....

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Autores principales: Koban, Florian, El-Kasaby, Ali, Häusler, Cornelia, Stockner, Thomas, Simbrunner, Benedikt M., Sitte, Harald H., Freissmuth, Michael, Sucic, Sonja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505579/
https://www.ncbi.nlm.nih.gov/pubmed/25869136
http://dx.doi.org/10.1074/jbc.M115.641357
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author Koban, Florian
El-Kasaby, Ali
Häusler, Cornelia
Stockner, Thomas
Simbrunner, Benedikt M.
Sitte, Harald H.
Freissmuth, Michael
Sucic, Sonja
author_facet Koban, Florian
El-Kasaby, Ali
Häusler, Cornelia
Stockner, Thomas
Simbrunner, Benedikt M.
Sitte, Harald H.
Freissmuth, Michael
Sucic, Sonja
author_sort Koban, Florian
collection PubMed
description The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr(603)–Thr(613)) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe(604), Ile(608), or Ile(612) by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu(615) at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu(615) and Arg(152). This interaction acts as a pivot in the conformational search associated with folding of SERT.
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spelling pubmed-45055792015-07-17 A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter Koban, Florian El-Kasaby, Ali Häusler, Cornelia Stockner, Thomas Simbrunner, Benedikt M. Sitte, Harald H. Freissmuth, Michael Sucic, Sonja J Biol Chem Cell Biology The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr(603)–Thr(613)) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe(604), Ile(608), or Ile(612) by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu(615) at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu(615) and Arg(152). This interaction acts as a pivot in the conformational search associated with folding of SERT. American Society for Biochemistry and Molecular Biology 2015-05-22 2015-04-13 /pmc/articles/PMC4505579/ /pubmed/25869136 http://dx.doi.org/10.1074/jbc.M115.641357 Text en © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license (http://creativecommons.org/licenses/by/3.0) .
spellingShingle Cell Biology
Koban, Florian
El-Kasaby, Ali
Häusler, Cornelia
Stockner, Thomas
Simbrunner, Benedikt M.
Sitte, Harald H.
Freissmuth, Michael
Sucic, Sonja
A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title_full A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title_fullStr A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title_full_unstemmed A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title_short A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter
title_sort salt bridge linking the first intracellular loop with the c terminus facilitates the folding of the serotonin transporter
topic Cell Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505579/
https://www.ncbi.nlm.nih.gov/pubmed/25869136
http://dx.doi.org/10.1074/jbc.M115.641357
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