Identify compounds that target post-Golgi secretion(RMI)

描述（由申请人提供）：细胞内转运和细胞表面和分泌蛋白的靶向递送是所有真核细胞的基本过程。从分泌细胞器的管腔和膜中招募货物蛋白，使膜变形以形成运输囊泡，并将囊泡融合到适当位置的机制已经被描述为几个运输步骤，并且在真核生物中被证明是很保守的。货物分拣和靶向的特殊性需要独特的调节和结构蛋白在每个运输步骤。酿酒酵母在鉴定和表征许多这些结构和调节蛋白方面具有不可估量的价值，并且是研究分泌过程的一个成熟的模型系统。然而，酵母和哺乳动物细胞中运输途径的分支使得对晚期（后高尔基体）分泌途径的研究变得复杂，而胞外货物分拣和从高尔基体和内体中退出所需的分子机制在很大程度上是未知的。由于货物可以从阻塞的路线中分离出来，并通过另一条路线分泌，因此仅在高尔基体的一条路线上有缺陷的突变体不会表现出容易筛选的分泌表型，因此难以分离。我们正在通过对一种突变酵母菌株进行筛选来解决这个问题，这种突变酵母菌株在两条已知的胞外途径中已经存在缺陷，因此干扰剩余的途径应该会导致容易检测到的完全分泌阻滞。我们已经成功地在经典遗传学（突变筛选）方法中使用了这种策略；然而，一种“化学遗传学”方法有望更有效地识别所需的基因，此外，还应该产生在治疗或作为研究工具中具有潜在用途的化合物。因此，我们建议使用高通量、基于细胞的化学文库筛选来鉴定特异性干扰胞外通路分支的化合物（以及随后的靶标）。

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.