Biogenesis of Transport Vesicles Coated by COPI

描述（由申请人提供）：阐明由外壳蛋白I （COPI）复合物调控的细胞内运输机制，我们发现gtase -激活蛋白（GAP）为小GTPase adp -核糖基化因子“！（ARF1）不仅作为ARF1的负调节因子，而且作为一种包被成分作为ARF1的效应因子。这一发现推翻了普遍认为GAP在COPI囊泡脱壳中起作用并应拮抗囊泡形成的观点，但与COPII囊泡形成的机制一致，其中相应的GAP也被证明是COPII包被复合物的一个组成部分。最近，我们对COPI囊泡重构系统进行了改进，发现这一过程需要辅助蛋白，这类似于网格蛋白囊泡的形成，已被证明需要多种辅助蛋白。因此，由于COPI转运的关键机制现在似乎比以前怀疑的更类似于对其他已明确表征的转运途径的阐明，这种在关键机制中观察到的守恒性也成为当前应用中寻求进一步阐明COPI囊泡形成和脱壳机制的总体驱动假设。首先，由于我们已经发现GAP不会直接触发COPI囊泡脱衣，我们将通过系统纯化方法确定预测的新型脱衣因子，并通过检查网格蛋白囊泡脱衣机制的潜在线索来补充这一策略。其次，正如我们最近发现的，Brefeldin-A adp -核糖基化底物（BARS）和亲内蛋白B作为COPI囊泡形成的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是如何实现这一作用的，这可能会再次被来自网格蛋白囊泡裂变机制的潜在线索所促进。第三，我们已经确定了磷脂酶D （PLD）活性在COPI囊泡形成中的新作用。因此，我们将澄清这一作用，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antiqutin（我们最近发现它与ARFGAP1相互作用）在COPI囊泡形成和/或货物分拣中是否以及可能如何发挥作用。我们预计，这些研究的完成不仅将进一步阐明COPI的运输机制，而且还将推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，由于细胞内运输途径的缺陷被认为是越来越多人类疾病的原因，我们的发现可能广泛适用于理解和治疗这些疾病。

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.