Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.

译文

携带荧光货物蛋白的分泌颗粒被广泛用于研究颗粒生物发生,成熟和调节胞吐作用。我们将可溶性分泌蛋白肽基甘氨酸 α-羟化单加氧酶 (PHM) 与绿色荧光蛋白 (GFP) 融合,以研究颗粒形成。当在AtT-20或GH3细胞中表达时,PHM-GFP融合蛋白从内源性激素 (促肾上腺皮质激素,生长激素) 分配到单独的分泌颗粒池中。外源和内源颗粒蛋白均被存储并释放以响应促分泌素。重要的是,我们发现内容物蛋白质的分离不是过度表达的产物,也不是GFP标记的蛋白质所特有的。管腔酸化和富含胆固醇的膜微域在可溶性蛋白分离中均未发挥重要作用。我们的数据表明,货物蛋白的内在生物物理特性决定了它们的差异分选,在颗粒成熟过程中发生了分离。可以自我聚集的蛋白质很可能会分成单独的颗粒,只能容纳几千个拷贝的任何含量的蛋白质; 缺乏三级结构的蛋白质更有可能均匀分布成分泌颗粒。因此,一个简单的 “自聚集默认” 理论可以解释自然存在的蛋白质和外源含量蛋白质彼此分离成不同的分泌颗粒的趋势,但鲜为人知但通常观察到。

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