An Amphipathic α-Helix Directs Palmitoylation of the Large Intracellular Loop of the Sodium/Calcium Exchanger

Fiona Plain, Samitha Dilini Congreve, Rachel Sue Zhen Yee, Jennifer Kennedy, Jacqueline Howie, Chien-Wen Kuo, Niall J. Fraser, William Fuller (Lead / Corresponding author)

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Abstract

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 (PIP2), and is facilitated by palmitoylation of a single cysteine at position 739 within NCX's large intracellular loop. 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 C739 in FL-NCX1 and deletions on the N-terminal side of C739 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 C739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate C739 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.
Original languageEnglish
Pages (from-to)10745-10752
Number of pages8
JournalJournal of Biological Chemistry
Volume292
Issue number25
Early online date21 Apr 2017
DOIs
Publication statusPublished - 23 Jun 2017

Keywords

  • sodium-calcium exchange
  • Protein acylation
  • Protein palmitoylation
  • Calcium transport
  • Sodium transport
  • Acyltransferase

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