An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger
| Autor: | Fiona Plain, Jennifer L. Kennedy, Jacqueline Howie, William Fuller, Rachel Sue Zhen Yee, Samitha Dilini Congreve, Niall J. Fraser, Chien-Wen Kuo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Lipoylation Mutation Missense Biochemistry protein palmitoylation Protein Structure Secondary Sodium-Calcium Exchanger 03 medical and health sciences Protein acylation Dogs Palmitoylation Protein Domains acyltransferase calcium transport Animals Humans Protein palmitoylation Editors' Picks Gap-43 protein sodium-calcium exchange protein acylation Molecular Biology Alanine chemistry.chemical_classification Sodium-calcium exchanger biology sodium transport Cell Biology Amino acid 030104 developmental biology HEK293 Cells chemistry Amino Acid Substitution biology.protein Biophysics cardiovascular system lipids (amino acids peptides and proteins) Protein Processing Post-Translational Intracellular |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | The electrogenic sodium/calcium exchanger (NCX) mediates bidirectional calcium transport controlled by the transmembrane sodium gradient. NCX inactivation occurs in the absence of phosphatidylinositol 4,5-bisphosphate and is facilitated by palmitoylation of a single cysteine at position 739 within the large intracellular loop of NCX. The aim of this investigation was to identify the structural determinants of NCX1 palmitoylation. Full-length NCX1 (FL-NCX1) and a YFP fusion protein of the NCX1 large intracellular loop (YFP-NCX1) were expressed in HEK cells. Single amino acid changes around Cys-739 in FL-NCX1 and deletions on the N-terminal side of Cys-739 in YFP-NCX1 did not affect NCX1 palmitoylation, with the exception of the rare human polymorphism S738F, which enhanced FL-NCX1 palmitoylation, and D741A, which modestly reduced it. In contrast, deletion of a 21-amino acid segment enriched in aromatic amino acids on the C-terminal side of Cys-739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate Cys-739 palmitoylation. Introduction of negatively charged amino acids to the hydrophobic face or of helix-breaking prolines impaired palmitoylation of both YFP-NCX1 and FL-NCX1. Alanine mutations on the hydrophilic face of the helix significantly reduced FL-NCX1 palmitoylation. Of note, when the helix-containing segment was introduced adjacent to cysteines that are not normally palmitoylated, they became palmitoylation sites. In conclusion, we have identified an amphipathic α-helix in the NCX1 large intracellular loop that controls NCX1 palmitoylation. NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence. |
| Databáze: | OpenAIRE |
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