The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3/WDR7 in neuroendocrine cells
The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3/WDR7 in neuroendocrine cells
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DOI：
10.1074/jbc.ra119.007504
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发表时间：
2019-06-14
影响因子：
4.8
通讯作者：
Martin, Thomas F. J.
中科院分区：
文献类型：
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作者：
Crummy, Ellen;Mani, Muralidharan;Martin, Thomas F. J.
液泡型 H+-ATP 酶 (V-ATP 酶) 有助于 pH 调节，并在分泌和内吞途径中发挥关键作用。神经内分泌细胞中的致密核心囊泡 (DCV) 维持在酸性 pH 值下，这是神经递质负载的电化学驱动力的一部分，也是激素前肽加工所需的。 CAPS1（又名钙依赖性分泌激活蛋白，CADPS）是 DCV 胞吐作用所需的囊泡结合启动因子，其基因缺失会消除 DCV 膜上的 pH 梯度并减少神经递质负荷。然而，CAPS1 与 DCV 结合及其调节囊泡 pH 的基础尚未确定。在这里，对脑膜组分中的 CAPS1 免疫沉淀物进行 MS 分析表明，CAPS1 与包含 Dmx 样 2 (DMXL2) 和 WD 重复结构域 7 (WDR7) 蛋白的 rabconnectin3 (Rbcn3) 复合物结合。使用免疫荧光显微镜，我们发现 Rbcn3/DMXL2 和 Rbcn3/WDR7 与人神经内分泌 (BON) 细胞 DCV 上的 CAPS1 共定位。 shRNA 介导的 Rbcn3/WDR7 敲低将 CAPS1 从 DCV 重新分配到细胞质，表明 Rbcn3/WDR7 对于 CAPS1 的最佳 DCV 定位至关重要。此外，无细胞实验揭示了 CAPS1 与 Rbcn3/WDR7 的直接结合，细胞分析表明 Rbcn3/WDR7 将可溶性 CAPS1 招募到膜上。正如报道的 Rbcn3 与 V-ATPase 关联所预期的那样，我们发现敲除神经内分泌细胞中的 CAPS1、Rbcn3 或 Rbcn3 会损害 DCV 再酸化的速率。这些发现揭示了 CAPS1 与 DCV 结合以及 CAPS1 通过 Rbcn3/WDR7 相互作用调节 V-ATP 酶活性的基础。
Vacuolar-type H+-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3/DMXL2 and Rbcn3/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3/WDR7, and cell assays indicated that Rbcn3/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3, or Rbcn3 in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3/WDR7 interactions.