Abstract：
Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.
摘要：
内分泌细胞利用分泌颗粒的受调节的胞吐作用来分泌激素和神经递质。分泌性颗粒胞吐取决于时空变量，如接近质膜和年龄，新产生的颗粒优先释放。尽管最近取得了进展，我们对胰岛素颗粒的分子组成和相关变化缺乏全面的认识。这里,我们报道了一种从胰岛素瘤INS-1细胞中纯化不同年龄胰岛素分泌颗粒的策略。用可切割的CLIP标签标记颗粒驻留蛋白phogrin，我们获得了年龄不同的颗粒的完整部分，用于蛋白质组学和脂质组学分析。我们发现脂质成分会随着时间的推移而变化，以及膜的物理性质，和驱动蛋白-1重链（KIF5b）以及Ras相关蛋白3a（RAB3a）优先与年轻颗粒缔合。Further,我们将RhoGTPase激活蛋白(ARHGAP1)鉴定为与胰岛素颗粒相关的胞浆因子.
