Identify compounds that target post-Golgi secretion(RMI)

描述（由申请人提供）：细胞表面和分泌蛋白的细胞内转运和靶向递送是所有真核细胞中的基本过程。 从分泌细胞器的腔和膜招募货物蛋白，使膜变形以形成运输囊泡，并将囊泡融合靶向到适当位置的机制已经描述了几个运输步骤，并显示在真核生物中很好地保守。 货物分选和靶向的特异性要求在每个运输步骤中具有独特的调节和结构蛋白。 酵母酿酒酵母在鉴定和表征许多这些结构和调节蛋白方面是非常宝贵的，并且是研究分泌过程的良好建立的模型系统。 然而，在酵母和哺乳动物细胞中的运输途径的分支使晚期（后高尔基体）分泌途径的研究变得复杂，并且外泌货物分选和从高尔基体和内体退出所需的分子机制在很大程度上是未知的。 由于货物可以从阻断的途径中分选出来并通过替代途径分泌，因此仅在高尔基体的一条途径中有缺陷的突变体不表现出容易筛选的分泌表型，因此难以分离。 我们正在接近这个问题，进行筛选与突变酵母菌株，已经有缺陷的两个已知的外排途径之一，使干扰其余的路线应该会导致一个完整的分泌块，很容易检测。 我们已经成功地使用这种策略在一个经典的遗传学（突变体筛选）的方法，然而，“化学遗传学”的方法有望更有效地确定所需的基因，此外，应该产生化学化合物与潜在的治疗用途或作为一种研究工具。 因此，我们建议使用高通量，基于细胞的化学文库筛选，以确定化合物（随后，他们的目标），特异性干扰一个分支的胞外途径。

DESCRIPTION (provided by applicant): The intracellular transport and targeted delivery of cell surface and secreted proteins is a fundamental process in all eukaryotic cells. The machinery that recruits cargo proteins from the lumen and membrane of secretory organelles, deforms the membrane for transport vesicle formation, and targets vesicle fusion to the appropriate location has been described for several transport steps and shown to be well conserved among eukaryotes. The specificity of cargo sorting and targeting demands unique regulatory and structural proteins at each transport step. The yeast Saccharomyces cereviseae has been invaluable in identifying and characterizing many of these structural and regulatory proteins and is a well-established model system in the study of the secretory process. However, the branching of transport routes in both yeast and mammalian cells has complicated studies of the late (post-Golgi) secretory pathway, and the molecular machinery required for exocytic cargo sorting and exit from the Golgi and endosomes is largely unknown. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route from the Golgi do not exhibit an easily screened secretory phenotype and are therefore difficult to isolate. We are approaching this problem by conducting screens with a mutant yeast strain that is already defective in one of the two known exocytic routes, so that interfering with the remaining route should cause a complete secretory block that is easy to detect. We have successfully used this strategy in a classical genetics (mutant screen) approach; however, a "chemical genetics" approach promises to be more efficient in identifying the desired genes an should in addition, yield chemical compounds with potential use in therapeutics or as a research tool. We therefore propose to use a high-throughput, cell-based screen of a chemical library to identify compounds (and subsequently, their targets) that interfere specifically with one branch of the exocytic pathway.