Carnell L, Moore H P
University of California, Department of Molecular and Cell Biology, Berkeley 94720-3200.
J Cell Biol. 1994 Nov;127(3):693-705. doi: 10.1083/jcb.127.3.693.
To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.
为深入了解蛋白质分选的调控机制，我们开发了一个系统，该系统可重建未成熟分泌颗粒的形成及其与质膜的融合过程。将半完整的PC12细胞与ATP和胞质溶胶一起孵育15分钟，以使未成熟颗粒形成，然后置于含有30微摩尔无钙[Ca2+]的缓冲液中以诱导胞吐作用。通过调节途径的转运，通过在反式高尔基体网络（TGN）中标记的分泌粒蛋白II（SgII）的释放来测定，受到ATP耗尽或加入100微摩尔GTPγS、50微摩尔AlF3-5或5微克/毫升布雷菲德菌素A（BFA）的抑制。当在未成熟颗粒形成后添加时，GTPγS刺激而非抑制胞吐作用。因此，该系统中未成熟颗粒的胞吐作用类似于完全成熟颗粒的特征。SgII通过调节途径的转运效率比糖胺聚糖链高四倍，这表明SgII在从TGN排出时在一定程度上进行了分选。添加A23187以从TGN释放Ca2+对SgII分选到未成熟颗粒中没有显著影响。相反，腔内钙的耗尽抑制了阿黑皮素原（POMC）和胰岛素原的内蛋白水解切割。这些结果确立了腔内Ca2+在激素原加工中的重要性，但提出了一个问题，即腔内钙对于SgII正确分选到未成熟颗粒中是否是必需的。用离子载体或弱碱破坏细胞器pH梯度会导致通过组成型和调节型途径的转运均受到抑制。
