GTP-BINDING PROTEINS AND INTRACELLULAR TRANSPORT

GTP 结合蛋白和细胞内转运

• 批准号: 3302460
• 负责人: PAUL R MELANCON
• 金额: $ 18.89万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302460
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; animal tissue; cell free system; eukaryote; gene expression; genetic library; guanine nucleotide binding protein; high performance liquid chromatography; immunoelectron microscopy; immunofluorescence technique; intracellular transport; laboratory mouse; laboratory rabbit; membrane proteins; messenger RNA; molecular cloning; monoclonal antibody; polymerase chain reaction; protein sequence; protein structure function; protein transport

The long-term objective of this project is to achieve a biochemical description of the mechanisms underlying protein traffic between membrane-bounded compartments of eukaryotic cells. Two distinct GTP- dependent Golgi Binding Factors, or GGBFs, were purified from tissue on the basis of their ability to cause GTPgammaS-dependent block of a cell- free assay that reconstitutes intra-Golgi transport. Both GGBFs are members of the ADP-ribosylation factor family of GTPases, but have different activities in the Golgi transport assay. The ARF family contains a large number of related proteins that may be involved in a wide range of cellular reactions. Most of our efforts will concentrate on elucidating the function of GGBFs in intra-Golgi transport and testing the possibility that each member of this family has a specialized function using both biochemical and genetic approaches. The specific aims of the project can be summarized as follows. (1) Further characterize GGBFs and related members of the ARF family using GTP-binding and cell-free transport assays in order establish if different ARFs have specialized function. (2) Identify GGBFs by microsequencing, clone their cDNAs and overexpress proteins in E. coli. (3) Raise polyclonal and monoclonal antibodies by immunization with pure protein or peptides derived from unique sequences of GGBFs. (4) Determine whether GGBFs are required to observe intra-Golgi transport and/or to produce vesicle intermediates. (5) Identify novel transport factors on the basis their effect on GGBF-activity and on the binding and hydrolysis of GTP by GGBFs (or on the basis of their association with GTP-bound GGBF). (6) Select "RNA antibodies" using SELEX as a way of circumventing current problems in obtaining specific and neutralizing antibodies to ARFs. Determine function(s) of GGBFs in vivo either by expressing high levels of inhibitory RNAs, or by reducing GGBF mRNA levels using antisense oligodeoxynucleotides. Members of the ARF family are likely to be involved in controlling assembly of components of the intracellular transport machinery. The work proposed in this application will lead to a more in-depth understanding of the function of these proteins. Furthermore, the identification and characterization of proteins with which they interact will contribute significantly towards a description of the mechanisms regulating intracellular traffic.

该项目的长期目标是实现生化 描述蛋白质之间的运输机制 真核细胞的膜界区室。 两种不同的 GTP- 从组织中纯化出依赖性高尔基体结合因子（GGBF） 它们引起细胞 GTPgammaS 依赖性阻断的能力的基础 重建高尔基体内运输的免费测定。 两个 GGBF 都是 GTPase 的 ADP-核糖基化因子家族的成员，但具有 高尔基体转运测定中的不同活性。 ARF家族 含有大量的相关蛋白质，这些蛋白质可能参与 广泛的细胞反应。 我们的大部分努力将集中在 阐明 GGBF 在高尔基体内运输和测试中的功能 这个家庭的每个成员都有专门的可能性 使用生化和遗传方法发挥功能。 该项目的具体目标可概括如下。 (1) 使用以下方法进一步表征 GGBF 和 ARF 家族的相关成员 GTP 结合和无细胞转运测定以确定是否 不同的ARF有专门的功能。 (2) 通过以下方式识别 GGBF 微测序、克隆其 cDNA 并在大肠杆菌中过表达蛋白质。 (3) 用纯疫苗免疫产生多克隆和单克隆抗体 源自 GGBF 独特序列的蛋白质或肽。 (4) 确定是否需要 GGBF 来观察高尔基体内的运输 和/或产生囊泡中间体。 (5) 识别新颖的运输方式 因素对 GGBF 活性和结合力的影响 GTP 被 GGBF 水解（或基于它们与 GTP 结合的 GGBF)。 (6) 使用SELEX选择“RNA抗体”作为方法 规避当前在获得具体和中和方面的问题 ARF 抗体。 通过以下方式确定体内 GGBF 的功能 表达高水平的抑制性 RNA，或通过减少 GGBF mRNA 使用反义寡脱氧核苷酸的水平。 ARF家族成员可能参与控制 细胞内运输机械部件的组装。 这 本申请中提出的工作将导致更深入的研究 了解这些蛋白质的功能。 此外， 与其相互作用的蛋白质的鉴定和表征 将对机制的描述做出重大贡献 调节细胞内交通。