Analyses on the secretory granule formation by using a yeast two hybrid system

颗粒蛋白(嗜铬颗粒蛋白/分泌颗粒蛋白)是一种酸性分泌蛋白，特异性地定位于各种内分泌细胞的分泌颗粒中。在大鼠垂体促性腺激素中，有代表性的两种颗粒物质--嗜铬颗粒A(CgA)和分泌颗粒II(SgII)分别定位于两个不同的分泌颗粒亚群，其大小和组成与这些颗粒的表达水平密切相关。基于这些发现，我们认为颗粒素是分泌颗粒的基本基质，但其靶向分泌颗粒的机制尚不清楚。在试图发现负责颗粒素向分泌颗粒转运的分子的过程中，颗粒蛋白家族的另一成员SgIII被鉴定为CGA的结合伙伴，并通过酵母双杂交筛选和检测。SgIII和CgA之间的相互作用在低pH和高钙条件下被增强，这种条件模拟了跨高尔基网络的流明环境。免疫细胞化学分析表明，这两种蛋白在体内共定位于内分泌细胞的分泌颗粒中，CgA的SgIII结合域的缺失削弱了其对AtT-20细胞分泌颗粒的靶向性。这些发现表明，SgIII在CgA和一个假定的分选受体之间起着界面分子的作用，该受体是为分泌颗粒而定的。这些由两个或两个以上颗粒组成的异嗜性复合体也可能通过为受调控的分泌蛋白和膜结合蛋白(如加工酶、离子通道、胺转运体和调控胞吐所需的融合机制)提供多个锚点来促进分泌颗粒的组织。

Granins (chromogranins/secretogranins) are acidic secretory proteins which are specifically localized to secretory granules in various endocrine cells with peptide hormones. As we previously demonstrated, two representative granins, chromogranin A (CgA) and secretogranin II (SgII), are separately localized to two distinct subsets of secretory granules in rat pituitary gonadotropes, and the sizes and composition of secretory granules in gonadotropes are closely associated with the expression levels of these granins. Based on these findings it is suggested that granins function as fundamental matrices of secretory granules, although the targeting mechanisms of the granins for secretory granules still remain unexplained.During attempts to discover the molecules responsible for the transport of granins to secretory granules, secretogranin III (SgIII), another member of the granin family, was identified as a binding partner of CgA with yeast two-hybrid screens and assays. The interaction between SgIII and CgA was augmented under low pH and high calcium conditions that mimic the luminal milieu at the trans-Golgi network. Immmunocytochemical analyzes demonstrated that both proteins were co-localized in secretory granules of endocrine cells in vivo , and that the deletion of the SgIII -binding domain of CgA impaired its targeting for secretory granules in AtT-20 cells. These findings suggest that SgIII acts as an interface molecule between CgA and a putative sorting receptor destined for secertory granules. These heterophilic complex of two or more granins might also contribute to the organization of secretory granules in general, by providing multiple anchor sites for both regulated secretory proteins and membrane-bound proteins such as processing enzymes, ion channels, amine transporters, and fusion machineries required for regulated exocytosis.