CHARACTERIZATION OF THE ARF FAMILY OF REGULATORY GTP-BINDING PROTEINS

ARF蛋白是一个结构和功能相似的亚家族， 保守的21 kDa GTP结合蛋白。 ARF现已被证明具有 在维护完整性和适当性方面的基本职能 内质网、高尔基复合体和核内体的功能 隔间 这些活动是下列行动的目标： 布雷菲德菌素A，一种正在开发中的真菌代谢物， 作为潜在抗肿瘤药物的发展治疗计划 在这个项目中描述的工作重点是表征 ARF家族的成员在序列、蜂窝位置和 功能协调发展的 我们已经发现，在人类中，这个家族不仅包括 5个以前鉴定的基因产物，但超过10个 结构相关基因及其产物。 我们从事克隆 并对这个新扩大的家族进行测序，开始记录其大小， 功能和重要性的细胞生理学和病理生理学。 在 此外，随着序列信息变得可用，我们继续 产生用于诊断目的的更特异的抗血清， 免疫定位 我们继续开发更简单的真核生物， 酵母，以确定ARF蛋白的新功能。 我们将执行 鉴定ARF突变的基因外抑制因子的功能筛选 在努力确定其他成分的ARF途径和潜在的 ARF活动的监管机构。 表达了大量的突变体， 酵母也是目前正在进行的类似研究的背景， 在培养的哺乳动物细胞中表达野生型和突变型ARF蛋白。 一个详尽的屏幕现在已经确定了至少一个温度 ARF 1的敏感等位基因，将用于帮助确定 ARF的功能、ARF空小区的后果以及功能 筛选ARF无效细胞的基因外抑制因子。 ARF的三维晶体结构也正在解决， 有助于确定关键功能域和最终的 特定抑制剂的设计。

The ARF proteins are a sub-family of structurally and functionally conserved 21 kDa GTP binding proteins. ARF has now been shown to have essential functions in the maintenance of the integrity and proper functioning of the endoplasmic reticulum, Golgi complex, and endosome compartments. These activities are the targets for the actions of Brefeldin A, a fungal metabolite under development in the the Developmental Therapeutics Program as a potential anti-neoplastic agent in man. Work described in this project focuses on characterizing the members of the ARF family with regard to sequence, cellular location, and functions. We have discovered that in humans this family consists not only of the five previously identified gene products, but of more than 10 structurally related genes and their products. We are engaged in cloning and sequencing this newly enlarged family to begin to document its size, functions and importance to cell physiology and pathophysiology. In addition, as sequence information becomes available we continue to produce more specific antisera for diagnostic purposes and immunolocalization. We have continued to exploit simpler eukaryotic organisms, particularly yeast, to identify new functions for ARF proteins. We will perform functional screens to identify extragenic suppressors of ARF mutations in efforts to identify other components of the ARF pathway and potential regulators of ARF activities. Expression of a number of mutants in yeast has also served as background for similar studies now underway to express wild-type and mutant ARF proteins in mammalian cells in culture. An exhaustive screen has now identified at least one temperature sensitive allele of ARF1 which will be used to assist in defining functions of ARF, consequences of an arf null cell, and functional screens for extragenic suppressors of the arf null cell. The three dimensional crystal structure of ARF is also being solved to aid in the determination of critical functional domains and ultimate design of specific inhibitors.