Proteins as functional elements of the machinery for the post-Golgi transport network

1)自噬：我们发现Apg 12-Apg 5缀合物与前体膜的结合是形成自噬体所必需的。此外，我们还发现该偶联物与其他蛋白质形成了一个大的复合物（约700 kDa）。我们纯化了复合物的一个组分，并测定了其氨基酸序列，它是一个63 kDa的新蛋白，在其N-末端区域与Apg 5结合。我们还表明，GATE 16和GABARAP，同源物的自噬体外周膜蛋白LC 3，以相同的方式处理LC 3和本地化的自噬体膜。最后，我们发现Apg 5缺陷的细胞中未折叠蛋白的积累加快，提示自噬参与了异常蛋白的排除。2）血小板的分泌：我们建立了一个体外重组系统，用于测定储存在致密核心颗粒中的5-羟色胺和储存在α颗粒中的血管性血友病因子的释放。它们的分泌依赖于ATP和胞浆。我们纯化了分泌所需的胞浆因子，并鉴定为PKCα。由于PKCα不足以重建释放，我们正试图确定释放所需的其他因素。

1) Autophagy : We found that binding of the Apg12-Apg5 conjugate to the precursor membranes is required for formation of autophagosomes. Furthermore, we showed that the conjugate forms a large complex (about 700kDa) together with other proteins. We purified one component of the complex and determined its amino acids sequence; it is a 63kDa novel protein, which binds to Apg5 at its N-terminal region. We also demonstrate that GATE 16 and GABARAP, homologues of the autophagosome peripheral membrane protein LC3, are processed in the same way as LC3 and localize to the autophagosomal membranes. Finally, we found that the unfolded protein accumulation is accelerated in the Apg5-deficient cells, suggesting that autophagy is involved in exclusion of abnormal proteins.2) Secretion in platelet : We established a in vitro reconstitution system for assaying release of serotonin stored in the dense-core granules and von Willebrand factor stored in the α granules by using the platelet whose plasma membrane are permeabilized by Streptolysin-O. Their secretion was dependent on ATP and cytosol. We purified the cytosolic factor required for the secretion and identified it as PKCα. Since PKCα was not sufficient to reconstitute the release, we are trying to identify other factors necessary for the release.