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

1)自噬：我们发现Apg12-Apg5偶联物与前体膜的结合是自噬体形成所必需的。此外，我们发现该偶联物与其他蛋白质形成一个大的复合物（约700kDa）。我们纯化了该复合物的一个组分并测定了其氨基酸序列；它是一个63kDa的新蛋白，在Apg5的n端区域结合。我们还证明，自噬体外周膜蛋白LC3的同源物GATE 16和GABARAP的加工方式与LC3相同，并定位于自噬体膜。最后，我们发现在apg5缺失的细胞中，未折叠蛋白的积累加速，这表明自噬参与了异常蛋白的排除。2)血小板分泌：我们建立了一种体外重建系统，以测定致密核颗粒中储存的5 -羟色胺和α颗粒中储存的血管性血友病因子的释放，该系统采用链溶菌素- o渗透质膜的血小板。它们的分泌依赖于ATP和细胞质。我们纯化了分泌所需的胞质因子，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.