Biogenesis of Transport Vesicles Coated by COPI

COPI 包被的运输囊泡的生物发生

• 批准号: 7211297
• 负责人: VICTOR W HSU
• 金额: $ 39.38万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7211297
• 关键词: ADP-Ribosylation Factor 1; ADP-Ribosylation Factors; ARFGAP1; Address; Affect; Automobile Driving; Biogenesis; Brefeldin A; Capsid Proteins; Classification; Clathrin; Coat Protein Complex I; Complement; Complex; Cytosol; Defect; Disease; GTPase-Activating Proteins; Intracellular Transport; Membrane; Monomeric GTP-Binding Proteins; Numbers; Pathway interactions; Phospholipase D; Phospholipid Metabolism; Play; Process; Protein Isoforms; Proteins; Role; Sorting - Cell Movement; System; Testing; Transport Vesicles; Vesicle; human disease; novel; polymerization; protein function; reconstitution

DESCRIPTION (provided by applicant): Elucidating mechanisms of intracellular transport regulated by the Coat Protein I (COPI) complex, we have found that the GTPase-activating protein (GAP) for the small GTPase ADP-Ribosylation Factor"! (ARF1) acts not only as a negative regulator of ARF1, but also as its effector by being a coat component. This finding reverses the prevailing view that the GAP functions in the uncoating of COPI vesicles and should antagonize vesicle formation, but is consistent with mechanisms elucidated for the formation of COPII vesicles, where the corresponding GAP has also been shown to function as a component of the COPII coat complex. Refining the COPI vesicle reconstitution system recently, we have revealed that this process requires accessory proteins, which is similar to the formation of clathrin vesicles that have been shown to use multiple accessory proteins. Thus, as key mechanisms of COPI transport now appear to be more similar to that elucidated for the other well-characterized transport pathways than previously suspected, this observed conservation in key mechanisms also becomes the overall driving hypothesis in the current application that seeks to further elucidate mechanisms of COPI vesicle formation and uncoating. First, as we have found that GAP does not directly trigger COPI vesicle uncoating, we will identify predicted novel uncoating factor(s) by a systematic purification approach and also complement this strategy by examining for potential clues from mechanisms of uncoating that are better elucidated for clathrin vesicles. Second, as we have found recently that Brefeldin-A ADP-Ribosylated Substrate (BARS) and endophilin B play interchangeable roles as key components of the fission machinery for COPI vesicle formation, we will determine more precisely how they achieve this role, which again will likely be facilitated by potential clues from mechanisms of fission that have been better elucidated for clathrin vesicles. Third, we have identified a novel role for phospholipase D (PLD) activity in COPI vesicle formation. Thus, we will clarify this role, which will also address a current controversy regarding the precise role of this activity in COPI vesicle formation. Fourth, we will examine whether and potentially how antiquitin, which we have shown recently to interact with ARFGAP1, plays a role in either COPI vesicle formation and/or cargo sorting. We anticipate that the completion of these studies will not only further elucidate mechanisms of COPI transport, but also advance a general understanding of vesicular transport, as key mechanisms are likely to be conserved across all transport pathways. Thus, as defects in intracellular transport pathways are being appreciated as the cause for a growing number of human diseases, our findings will likely be broadly applicable to understanding and treating these diseases.

描述（由申请人提供）：为了阐明外壳蛋白 I (COPI) 复合物调节的细胞内转运机制，我们发现小 GTP 酶 ADP-核糖基化因子 (ARF1) 的 GTP 酶激活蛋白 (GAP) 不发挥作用。不仅作为 ARF1 的负调节因子，而且作为涂层成分也作为其效应器。这一发现推翻了 GAP 在 COPI 脱涂层中发挥作用的普遍观点。囊泡并应拮抗囊泡形成，但与已阐明的 COPII 囊泡形成机制一致，其中相应的 GAP 也已被证明可作为 COPII 外壳复合物的组成部分。该过程需要辅助蛋白，这类似于网格蛋白囊泡的形成，已被证明使用多种辅助蛋白作为关键机制。 COPI 运输的机制现在似乎与之前怀疑的其他明确表征的运输途径所阐明的更为相似，这种观察到的关键机制的保守性也成为当前申请中的总体驱动假设，旨在进一步阐明 COPI 囊泡形成的机制和脱漆。首先，由于我们发现 GAP 不会直接触发 COPI 囊泡脱壳，因此我们将通过系统纯化方法识别预测的新型脱壳因子，并通过检查脱壳机制中的潜在线索来补充这一策略，这些线索可以更好地阐明网格蛋白囊泡。其次，正如我们最近发现的那样，布雷菲德菌素 A ADP 核糖基化底物 (BARS) 和内亲素 B 作为 COPI 囊泡形成裂变机制的关键组成部分发挥着可互换的作用，我们将更准确地确定它们如何实现这一作用，这将再次网格蛋白囊泡的裂变机制的潜在线索可能会促进这一过程，而这些裂变机制已得到更好的阐明。第三，我们发现了磷脂酶 D (PLD) 活性在 COPI 囊泡形成中的新作用。因此，我们将阐明这一作用，这也将解决当前关于该活性在 COPI 囊泡形成中的确切作用的争议。第四，我们将研究古蛋白（我们最近证明其与 ARFGAP1 相互作用）是否以及可能如何在 COPI 囊泡形成和/或货物分选中发挥作用。我们预计这些研究的完成不仅将进一步阐明 COPI 运输机制，而且还将促进对囊泡运输的一般理解，因为关键机制可能在所有运输途径中都是保守的。因此，随着细胞内转运途径的缺陷被认为是越来越多的人类疾病的原因，我们的发现可能广泛适用于理解和治疗这些疾病。