Fiche publication
Date publication
septembre 2019
Journal
The Journal of biological chemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr KLYMCHENKO Andrey
Tous les auteurs :
Jiang M, Hu J, White FKH, Williamson J, Klymchenko AS, Murthy A, Workman SW, Tseng GN
Lien Pubmed
Résumé
Junctophilins (JPH1-JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM). These ER/SR-PM junctions bring Ca-release channels in the ER/SR and Ca as well as Ca-activated K channels in the PM to within 10-25 nm. Such proximity is critical for excitation-contraction coupling in muscles, Ca modulation of excitability in neurons, and Ca homeostasis in nonexcitable cells. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD). Their N-terminal embrane-ccupation-ecognition-exus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH-PM binding is not clear. We investigate whether -palmitoylation of cysteine (Cys), a critical mechanism controlling peripheral protein binding to PM, occurs in JPHs. We focus on JPH2 that has four Cys residues: three flanking the MORN motifs and one in the TMD. Using palmitate-alkyne labeling, Cu(I)-catalyzed alkyne-azide cycloaddition reaction with azide-conjugated biotin, immunoblotting, proximity-ligation-amplification, and various imaging techniques, we show that JPH2 is -palmitoylatable, and palmitoylation is essential for its ER/SR-PM tether function. Palmitoylated JPH2 binds to lipid-raft domains in PM, whereas palmitoylation of TMD-located Cys stabilizes JPH2's anchor in the ER/SR membrane. Binding to lipid-raft domains protects JPH2 from depalmitoylation. Unpalmitoylated JPH2 is largely excluded from lipid rafts and loses the ability to form stable ER/SR-PM junctions. In adult ventricular myocytes, native JPH2 is -palmitoylatable, and palmitoylated JPH2 forms distinct PM puncta. Sequence alignment reveals that the palmitoylatable Cys residues in JPH2 are conserved in other JPHs, suggesting that palmitoylation may also enhance ER/SR-PM tethering by these proteins.
Mots clés
ER–PM junction, endoplasmic reticulum (ER), excitation–contraction coupling (E-C coupling), lipid raft, palmitoylation, plasma membrane, post-translational modification
Référence
J. Biol. Chem.. 2019 Sep 6;294(36):13487-13501
