Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.

中文翻译：

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SLC9A7相互作用组的鉴定和生化特征。

细胞器和细胞质的pH稳态对大多数细胞过程至关重要，包括囊泡运输，蛋白质的翻译后修饰/加工以及受体-配体相互作用。SLC9A7（NHE7）被确定为独特的（Na +，K +）/ H +交换剂，可在反高尔基网络（TGN），内体和质膜之间动态循环。在这里，我们已经使用质谱技术来探索NHE7的亲和力捕获的相互作用组，从而鉴定出细胞骨架蛋白，细胞粘附分子，膜转运蛋白和信号分子。在这些结合蛋白中，钙钙调蛋白而不是脱钙钙调蛋白与NHE7结合并调节细胞器转运蛋白的活性。波形蛋白与人乳腺癌MDA-MB-231细胞中的内源性NHE7蛋白共免疫沉淀。相当数量的NHE7群体在迁移细胞中重新定位为聚焦复合物，并在聚焦复合物中显示与波形蛋白和肌动蛋白的共定位。在鉴定出的NHE7结合蛋白中，透明质酸盐和其他配体的细胞表面糖蛋白受体CD44显示与NHE7的相互作用受到调节。用佛波醇酯预处理细胞有助于NHE7-CD44相互作用和CD44的脂筏缔合。当脂质筏被化学破坏时，NHE7-CD44相互作用显着降低。这些结果表明NHE7在细胞表面隔室和细胞表面膜亚域中的潜在双重作用。透明质酸和其他配体的细胞表面糖蛋白受体显示出与NHE7相互作用的调控。用佛波醇酯预处理细胞有助于NHE7-CD44相互作用和CD44的脂筏缔合。当脂质筏被化学破坏时，NHE7-CD44相互作用显着降低。这些结果表明NHE7在细胞表面隔室和细胞表面膜亚域中的潜在双重作用。透明质酸和其他配体的细胞表面糖蛋白受体显示出与NHE7相互作用的调控。用佛波醇酯预处理细胞有助于NHE7-CD44相互作用和CD44的脂筏缔合。当脂质筏被化学破坏时，NHE7-CD44相互作用显着降低。这些结果表明NHE7在细胞表面隔室和细胞表面膜亚域中的潜在双重作用。