Colomer V, Kicska G A, Rindler M J
Department of Cell Biology, New York University Medical Center, New York 10016, USA.
J Biol Chem. 1996 Jan 5;271(1):48-55. doi: 10.1074/jbc.271.1.48.
A major unresolved issue in the field of secretory granule biogenesis is the extent to which the aggregation of granule content proteins is responsible for the sorting of regulated from constitutively secreted proteins. The aggregation process is postulated to take place in the trans-Golgi network and immature secretory granules as the proteins encounter mildly acidic pH and high calcium concentrations. We have developed in vitro assays that reconstitute the precipitation out of solution of secretory granule content proteins of anterior pituitary gland and adrenal medulla. In the assays, all of the major granule content polypeptides form a precipitate as the pH is titrated below 6.5, and this precipitate can be recovered in the pellet fraction after centrifugation. Addition of calcium is required for the aggregation of chromaffin granule content. In contrast to the proteins secreted by the regulated pathway, the constitutively secreted proteins IgG, albumin, and angiotensinogen, when added to the assays, remain predominantly in the supernatant. Among the individual proteins tested, prolactin is found to aggregate homophilically under these conditions and can drive the co-aggregation of other proteins, such as the chromogranins. Soluble forms of granule membrane proteins, including dopamine beta-hydroxylase and peptidyl glycine alpha-amidating enzyme also co-aggregated with granule content proteins. The results are consistent with the idea that spontaneous aggregation of proteins occurring under ionic conditions similar to those at the sites of granule formation is a property restricted to those proteins packaged in secretory granules. In addition, the association of luminal domains of membrane proteins with content proteins in vitro raises the possibility that analogous interactions between membrane-bound and content proteins also occur during granule formation in intact cells.
分泌颗粒生物发生领域一个主要未解决的问题是，颗粒内容物蛋白质的聚集在多大程度上导致了受调控分泌蛋白与组成型分泌蛋白的分选。据推测，随着蛋白质遇到轻度酸性pH值和高钙浓度，聚集过程发生在反式高尔基体网络和未成熟分泌颗粒中。我们已经开发了体外分析方法，可重构垂体前叶和肾上腺髓质分泌颗粒内容物蛋白质从溶液中沉淀的过程。在这些分析中，当pH值滴定到6.5以下时，所有主要的颗粒内容多肽都会形成沉淀，并且该沉淀在离心后可在沉淀部分中回收。嗜铬颗粒内容物的聚集需要添加钙。与受调控途径分泌的蛋白质相反，组成型分泌的蛋白质IgG、白蛋白和血管紧张素原，添加到分析中时，主要仍留在上清液中。在所测试的单个蛋白质中，发现催乳素在这些条件下会发生同源聚集，并能驱动其他蛋白质（如嗜铬粒蛋白）的共聚集。颗粒膜蛋白的可溶性形式，包括多巴胺β-羟化酶和肽基甘氨酸α-酰胺化酶，也与颗粒内容物蛋白共聚集。这些结果与以下观点一致，即在类似于颗粒形成部位的离子条件下发生的蛋白质自发聚集是包装在分泌颗粒中的蛋白质所特有的性质。此外，膜蛋白腔结构域与内容物蛋白在体外的结合增加了在完整细胞颗粒形成过程中膜结合蛋白与内容物蛋白之间也发生类似相互作用的可能性。
