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

颗粒蛋白（chromogranins/secretogranins）是一种酸性分泌蛋白，专门定位于各种内分泌细胞中具有肽激素的分泌颗粒。正如我们之前所证明的，在大鼠垂体促性腺激素中，两种具有代表性的颗粒，嗜色颗粒蛋白A （CgA）和分泌颗粒蛋白II （SgII）分别定位于两个不同的分泌颗粒亚群，促性腺激素中分泌颗粒的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，我们认为颗粒是分泌颗粒的基本基质，尽管颗粒对分泌颗粒的靶向机制仍未解释。在试图发现负责颗粒转运到分泌颗粒的分子的过程中，通过酵母双杂交筛选和测定，发现了颗粒蛋白家族的另一成员分泌颗粒蛋白III （SgIII）是CgA的结合伙伴。在低pH和高钙条件下，SgIII和CgA之间的相互作用增强，模拟了反式高尔基网络的腔内环境。免疫细胞化学分析表明，这两种蛋白在体内内分泌细胞的分泌颗粒中共定位，CgA的SgIII结合域的缺失破坏了其对at -20细胞分泌颗粒的靶向作用。这些发现表明，SgIII是CgA和一种被推测为分泌颗粒的分选受体之间的界面分子。这些由两个或多个颗粒组成的异亲性复合体也可能有助于分泌颗粒的组织，通过为受调节的分泌蛋白和膜结合蛋白（如加工酶、离子通道、胺转运蛋白和受调节的胞外分泌所需的融合机制）提供多个锚定位点。

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.