S-Palmitoylation of junctophilin-2 is critical for its role in tethering the sarcoplasmic reticulum to the plasma membrane
S-Palmitoylation of junctophilin-2 is critical for its role in tethering the sarcoplasmic reticulum to the plasma membrane
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DOI：
10.1074/jbc.ra118.006772
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发表时间：
2019-09-06
影响因子：
4.8
通讯作者：
Tseng, Gea-Ny
中科院分区：
文献类型：
--
作者：
Jiang, Min;Hu, Junping;Tseng, Gea-Ny
嗜连接蛋白（JPH 1-JPH 4）在可兴奋和不可兴奋细胞中表达，在那里它们束缚内质网/肌浆网（ER/SR）和质膜（PM）。这些ER/SR-PM连接将ER/SR中的Ca释放通道和PM中的Ca以及Ca激活的K通道带到10-25 nm内。这种接近对于肌肉中的兴奋-收缩偶联、神经元中兴奋性的钙调节以及非兴奋细胞中的钙稳态是至关重要的。JPH通过C-末端跨膜结构域（TMD）锚定在ER/SR中。它们的N-末端膜占据-消除-连接（MORN）基序可以结合磷脂。单独的MORN基序是否足以稳定JPH-PM结合尚不清楚。我们调查是否S-棕榈酰化的半胱氨酸（Cys），一个关键的机制控制外周蛋白结合PM，发生在JPH。我们专注于JPH 2，有四个半胱氨酸残基：三个侧翼的MORN基序和一个在TMD。使用棕榈酸-炔标记，Cu（I）催化的炔-叠氮环加成反应与叠氮共轭生物素，免疫印迹，邻近连接扩增，和各种成像技术，我们表明，JPH 2是S-棕榈酰化，棕榈酰化是必不可少的ER/SR-PM系链功能。棕榈酰化的JPH 2与PM中的脂筏结构域结合，而TMD定位的Cys的棕榈酰化使JPH 2在ER/SR膜中的锚稳定。与脂筏结构域的结合保护JPH 2免于脱棕榈酰化。未棕榈酰化的JPH 2在很大程度上被排除在脂筏之外，并失去形成稳定的ER/SR-PM连接的能力。在成年心室肌细胞中，天然JPH 2是S-棕榈酰化的，棕榈酰化的JPH 2形成不同的PM斑点。序列比对显示，棕榈酰化的半胱氨酸残基在JPH 2中是保守的，在其他JPH，这表明棕榈酰化也可能增强ER/SR-PM栓系这些蛋白质。
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.