COPI Transport

描述（由申请人提供）：囊泡运输在细胞内不同的细胞器间运送蛋白质和膜。衣壳蛋白是启动囊泡形成的核心机制。外壳蛋白I （COPI）是研究这一过程的一个范例，并被证明受小GTPase ADP-核糖基化因子1 （ARF1）的调节。在上一个资助期内，我们在了解依赖copi的运输方面取得了重大进展。首先，我们注意到细胞内运输现在也被认为是通过管状运输发生的，但负责小管形成的核心机制尚不清楚。为了解决这个问题，我们发现COPI复合体也启动小管的形成，这参与了促进顺行高尔基内运输和高尔基带的形成。因此，我们建议在当前的更新中确定预测在COPI小管与靶室对接中起作用的系绳，并确定货物如何在这些小管中运输。其次，我们注意到使ARF1失活的gtpase激活蛋白（GAP） ARFGAP1先前已被发现具有新的作用，也可作为外壳成分。然而，关于这一作用一直存在争议，特别是使用脂质体而不是高尔基膜的COPI重构研究。因此，我们建议阐明为什么脂质体为基础的系统不能重建ARFGAP1的外壳功能。此外，我们将阐明该GAP对ARFGAP2/3的作用，这两个成员关系密切。第三，我们最近发现了一种涉及脱氢酶反应的调节COPI囊泡形成的新机制，并建议进一步剖析这种调节是如何实现的。第四，我们最近也阐明了一种关键脂质磷脂酸（PA）在COPI囊泡裂变中起作用的复杂性。因此，我们也将寻求对PA在这一过程中如何起作用的更好的机制理解。总之，这些拟议的研究可能不仅会促进对COPI运输的机制理解，而且还会促进强调细胞内运输的一般原则。此外，由于这一基本过程对许多细胞事件至关重要，我们的研究结果也可能为多种人类疾病的发生提供新的分子见解。

DESCRIPTION (provided by applicant): Vesicular transport shuttles proteins and membranes among the different organellar compartments within the cell. Coat proteins act as the core machinery that initiates vesicle formation. Coat Protein I (COPI) has been a paradigm to studying this process, and has been shown to be regulated by the small GTPase ADP- Ribosylation Factor 1 (ARF1). In the last funding period, we have made significant advancements in understanding COPI-dependent transport. First, we note that intracellular transport is now also appreciated to occur through tubular transport, but the core machinery responsible for tubule formation has been unknown. Addressing this issue, we have found that the COPI complex also initiates tubule formation, which is involved in promoting anterograde intra-Golgi transport and Golgi ribbon formation. As such, we propose in the current renewal to identify tethers predicted to act in the docking of COPI tubules to target compartment, and also determine how cargoes are transported in these tubules. Second, we note that the GTPase-activating protein (GAP) that deactivates ARF1, ARFGAP1, has been found previously to have a novel role, acting also as a coat component. However, there has been continuing controversy regarding this role, in particular from COPI reconstitution studies that have used liposomes rather than Golgi membrane. Thus, we propose to elucidate why the liposome-based system has not been able to reconstitute the coat function of ARFGAP1. Moreover, we will clarify the role of this GAP with respect to ARFGAP2/3, which are two closely related members. Third, we have recently identified a novel mechanism of regulating COPI vesicle formation that involves a dehydrogenase reaction, and propose to further dissect out how this regulation is achieved. Fourth, we have also recently elucidated the complexity by which a critical lipid, phosphatidic acid (PA), acts in COPI vesicle fission. Thus, we will also seek a better mechanistic understanding of how PA acts in this process. Altogether, these proposed investigations will likely advance not only a mechanistic understanding of COPI transport, but also general principles that underline intracellular transport. Moreover, because this basic process is critical for many cellular events, our results may also suggest new molecular insights into how multiple human diseases occur. PUBLIC HEALTH RELEVANCE: We study how proteins and membrane are transported within the cell. We anticipate that our proposed studies will advance a fundamental understanding of this transport. Moreover, because this process is critical for multiple aspects of cellular function, we further anticipate in shedding key molecular insights into pathologic events that result in human diseases.