An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger

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 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.

Spatial organization of palmitoyl acyl transferases governs substrate localization and function

“…Although there is clear evidence that PAT-substrate specificity is partially governed by the amino acid sequence flanking the palmitoylated cysteine (Lemonidis et al, 2014(Lemonidis et al, , 2015(Lemonidis et al, , 2017Plain et al, 2017), some data argues a stochastic palmitoylation model, in which palmitoylation is governed by the accessibility of cysteine residues of membrane proteins located at a certain depth in the inner leaflet of the membrane, rather than by the local sequence motif (Rodenburg et al, 2017). The study demonstrates that genetically introduced cysteine residues in membrane proteins, even in prokaryotic membrane proteins, which are not usually palmitoylated, undergo generic palmitoylation so long as the cysteine residue is accessible to the catalytic center of the PAT, suggesting a lack of specificity for S-palmitoylation (Rodenburg et al, 2017).…”

Understanding the rules governing NCX1 palmitoylation

“…4,5 There are multiple cysteines in the NCX1 regulatory loop but only cysteine 739, located at the C-terminal end of this loop close to transmembrane domain 6, becomes palmitoylated. In our recent paper 6 we asked why this particular cysteine gets palmitoylated when neighboring ones do not. A fusion protein between the NCX1 regulatory loop and YFP is palmitoylated at C739 4 , unequivocally demonstrating that the exchanger's transmembrane domains are dispensable for palmitoylation.…”

How a helix imposes palmitoylation of a membrane protein: What one can learn from NCX

“…These results underscore the fundamental role of the ␣-helix in mediating recognition with one or more S-acylation enzymes and suggest that the principles of a "consensus sequence" may be largely inapplicable for recognizing the palmitoylation site in NCX (and perhaps in many other proteins as well). Indeed, other data in the paper from Fuller and colleagues (2) suggest that there are additional elements that influence palmitoylation: The first two turns of the ␣-helix are essential for recognition in both full-length and truncated NCX1 constructs, but the lack of the next two turns can be somehow mitigated by transmembrane domains in the full-length protein that likely help to position Cys-739 near the membrane. It will be exciting to identify the exact structural elements determining the interaction of the ␣-helix domain with the active site of S-acylation enzymes by applying advanced approaches of structural biology and biophysics.…”

“…A new study by Fuller and colleagues (2) provides a possible explanation for this conundrum in their discovery that a secondary structure is needed for palmitoylation of the sodium/calcium exchanger 1 (NCX1). 2 In mammals, there are three gene isoforms of NCX (NCX1-3), with their splice variants being expressed in a tissue-specific manner to maintain ion homeostasis (3). NCX proteins include 10 transmembrane helices (TM1-TM10) (4), with an extended cytoplasmic f-loop between TM5 and TM6 containing two Ca 2ϩ -binding regulatory domains (CBD1 and CBD2) connected through a very short interdomain linker ( Fig.…”