Formation of the Regulated Secretory Pathway: a View from the Cytosol

摘要 释放信号分子的能力，如肽激素、神经肽和许多生长 对适当的细胞外刺激作出反应的因子，对生理、行为和 发展。调节这种调节分泌的分泌小泡称为大致密核心小泡。 (LDCV)。它们从跨高尔基网络(TGN)发芽，在那里它们的可溶性货物聚集形成致密的 核心，而是细胞机制，特别是产生这些分泌物的细胞质机械 对囊泡的了解还不是很清楚。最近发表的对线虫的研究发现，HID-1是一种 胞浆因子参与神经肽的分选，但其功能尚不清楚。HID-1是外围设备 与TGN相关的膜蛋白及其表达仅限于神经内分泌细胞，提示 这可能有助于LDCV的生物发生。我们初步的大鼠功能丧失实验 基因组编辑产生的神经内分泌HID-1基因敲除(KO)细胞证实HID-1确实是 哺乳动物神经内分泌所必需的。我们现在将1)确定HID-1对LDCV的影响 使用生化和成像分析的组合形成，2)确定HID-1靶标如何特异性 3)确定调节HID-1与TGN可逆结合的机制，并评估其 对LDCV的形成具有重要意义。这些研究将提供关于细胞生物学和 一种新的参与膜转运的新的外周膜蛋白的生化 对我们理解神经内分泌的启示。

Abstract The ability to release signaling molecules, such as peptide hormones, neuropeptides, and many growth factors, in response to an appropriate extracellular stimulus, is central to physiology, behavior, and development. The secretory vesicles mediating this regulated secretion are called large dense core vesicles (LDCVs). They bud from the trans-Golgi network (TGN) where their soluble cargo aggregates to form a dense core, but the cellular mechanisms, and in particular, the cytosolic machinery that produces these secretory vesicles are still not well understood. Recent published studies in C. Elegans have identified HID-1 as a cytosolic factor involved in neuropeptide sorting, but its function remains unknown. HID-1 is a peripheral membrane protein associated with the TGN and its expression is restricted to neuroendocrine cells, suggesting that it might contribute to LDCV biogenesis. Our preliminary loss-of-function experiments with rat neuroendocrine HID-1 knockout (KO) cells generated by genome-editing have confirmed that HID-1 is indeed required for mammalian neuroendocrine secretion. We will now 1) determine how HID-1 contributes to LDCV formation using a combination of biochemical and imaging assays, 2) establish how HID-1 targets specifically to the TGN, and 3) identify the mechanism regulating the reversible binding of HID-1 to the TGN and assess its significance for LDCV formation. These studies will provide invaluable information about the cell biology and biochemistry of a novel peripheral membrane protein involved in membrane trafficking with important implications for our understanding of neuroendocrine secretion.