Abstract
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 Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH-PM binding is not clear. We investigate whether S-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 S-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 S-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.
Highlights
Junctophilins (JPH1–JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM)
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 S-palmitoylatable, and palmitoylation is essential for its ER/SR–PM tether function
1) JPH2 was S-palmitoylatable, both as heterologously expressed protein encoded by human JPH2 in COS-7 cells (Fig. 1, A and B), and as native protein in adult rat ventricular myocytes (Fig. 7B). 2) Palmitoylation is essential for JPH2’s role as an Endoplasmic reticulum–plasma membrane (ER–PM) tether (Fig. 5). 3) All four Cys side chains in human JPH2 were involved in S-palmitoylation (Fig. 1C). 4) Functionally, palmitoylation promoted JPH2 binding to lipid–raft domains of PM (Fig. 3), which likely involved Cys side chains flanking the MORN motifs based on their proximity to the PM
Summary
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. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD) Their N-terminal Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. We investigate whether S-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. Similar to other members of the junctophilin family, JPH2 is anchored in the ER/SR membrane by its C-terminal transmembrane domains (TMDs) and binds PM with its N-terminal Membrane Occupation and Recognition Nexus (MORN) motifs (MORN1–MORN8) [3]. We applied these approaches to study native JPH2 in adult ventricular myocytes
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