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，其特异性地定位于具有肽激素的各种内分泌细胞中的分泌颗粒。正如我们以前所证明的，两个代表性的颗粒，嗜铬颗粒蛋白A（CgA）和分泌颗粒蛋白II（SgII），分别定位于两个不同的子集的分泌颗粒在大鼠垂体促性腺激素细胞，和分泌颗粒的大小和组成与这些颗粒的表达水平密切相关。颗粒蛋白是分泌颗粒的基本基质，但颗粒蛋白的靶向机制尚不清楚。在寻找颗粒蛋白转运至分泌颗粒的分子的过程中，颗粒蛋白家族的另一成员分泌颗粒蛋白III（Sg III），通过酵母双杂交筛选和分析，鉴定为CgA的结合伴侣。SgIII和CgA之间的相互作用增强低pH值和高钙的条件下，模仿腔环境中的trans-Golgi网络。免疫细胞化学分析表明，这两种蛋白质共定位于体内内分泌细胞的分泌颗粒中，并且CgA的SgIII结合结构域的缺失损害了其针对AtT-20细胞中分泌颗粒的靶向。这些研究结果表明，SgIII作为CgA和一个假定的分选受体注定分泌颗粒之间的界面分子。这些两种或多种颗粒蛋白的嗜异性复合物也可能有助于分泌颗粒的组织，一般通过提供多个锚位点的调节分泌蛋白和膜结合蛋白，如加工酶，离子通道，胺转运蛋白，和融合机制所需的调节胞吐。

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.