Biogenesis of Transport Vesicles Coated by COPI

说明（由申请人提供）：阐明了由外壳蛋白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"的GTP酶激活蛋白（GAP）!（ARF1）不仅作为ARF1的负调节剂，而且作为其效应物通过作为包衣组分而起作用。这一发现推翻了普遍的观点，即GAP在COPI囊泡的脱壳中起作用，应该拮抗囊泡的形成，但与COPII囊泡形成的机制一致，其中相应的GAP也被证明是COPII外壳复合物的一种组分。最近，我们对COPI囊泡重构系统进行了改进，发现该过程需要辅助蛋白，这类似于网格蛋白囊泡的形成，网格蛋白囊泡已被证明使用多种辅助蛋白。因此，由于COPI转运的关键机制现在看起来比以前怀疑的更类似于针对其他充分表征的转运途径阐明的机制，因此在关键机制中观察到的这种保守性也成为本申请中的总体驱动假设，其寻求进一步阐明COPI囊泡形成和脱壳的机制。首先，由于我们已经发现GAP不直接触发COPI囊泡脱壳，我们将通过系统纯化方法鉴定预测的新脱壳因子，并通过检查来自网格蛋白囊泡更好阐明的脱壳机制的潜在线索来补充该策略。其次，我们最近发现布雷菲德蛋白-A ADP-核糖基化底物（BARS）和内亲素B作为COPI囊泡形成的裂变机制的关键组分发挥可互换的作用，我们将更精确地确定它们如何实现这一作用，这可能再次通过来自裂变机制的潜在线索来促进，这些裂变机制已被更好地阐明用于网格蛋白囊泡。第三，我们已经确定了一个新的作用，磷脂酶D（PLD）的活动在COPI囊泡的形成。因此，我们将澄清这一作用，这也将解决目前的争议，关于确切的作用，这一活动在COPI囊泡形成。第四，我们将研究antiquitin，我们最近已经表明，与ARFGAP 1相互作用，是否以及如何发挥作用，无论是COPI囊泡的形成和/或货物分选。我们预计，这些研究的完成不仅将进一步阐明COPI运输的机制，而且还推进了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。

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.