Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1

Caglar Gök,Alice Main,Xing Gao,Zsombor Kerekes,Fiona Plain,Chien-Wen Kuo,Alan D Robertson,Niall J Fraser,William Fuller

doi:10.1016/j.ceca.2021.102408

Caglar Gök, Alice Main + Show 7 more

Open Access

https://doi.org/10.1016/j.ceca.2021.102408

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Journal: Cell Calcium	Publication Date: Apr 8, 2021
Citations: 12	License type: cc-by

Affiliation: University of Glasgow, University of Dundee

Abstract

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740–756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery.

Highlights

The cardiac Na+/Ca2+ exchanger (NCX1) regulates cytosolic Ca2+ levels in myocytes and cardiac contractility by controlling the bidirectional transport of Na+ and Ca2+ ions across the surface mem brane
We found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-protein acyl-transferases (PATs), (3) NCX1 is still pal mitoylated when it is arrested in either Golgi or Endoplasmic Reticulum (ER), indicating that NCX1 is a substrate for multiple zDHHCPATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcel lular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER
It is notable that the close proximity of CBD2 (502–690) to the XIP binding (709–720 [14]) and palmitoylation (C739) sites means that the conformational flexibility of the entire C terminal half of the NCX1 regulatory loop may change according to Ca occupancy of CBD2, whether XIP is sequestered by PIP2, and whether C739 is palmitoylated [7]

Summary

Introduction

The cardiac Na+/Ca2+ exchanger (NCX1) regulates cytosolic Ca2+ levels in myocytes and cardiac contractility by controlling the bidirectional transport of Na+ and Ca2+ ions across the surface mem brane. The endogenous XIP domain, at the N terminal end of the same regulatory loop (Fig. 1A), inhibits NCX1 [10] but is usually sequestered by the phospholipid PIP2 [11]. Reversible palmitoylation is a key regulator of NCX1 activity [12,13] because it sensitizes NCX1 to XIP, and to PIP2, leading to enhanced NCX1-mediated trans membrane Ca flux when NCX1 is non-palmitoylated [14]. This effect of palmitoylation on NCX1 inactivation occurs without an apparent impact on the ability of calcium to activate NCX1 [15]. Understanding the cellular control of NCX1 palmitoylation is a high priority

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