COPI Transport

描述(申请人提供)：细胞内不同细胞器间的囊泡运输、蛋白质和膜。外壳蛋白是启动囊泡形成的核心机制。外壳蛋白I(COPI)是研究这一过程的典范，并被证明受小GTP酶ADP-核糖化因子1(ARF1)的调控。在上一个资助期，我们在理解依赖于COPI的运输方面取得了重大进展。首先，我们注意到细胞内转运现在也被认为是通过小管运输发生的，但负责小管形成的核心机制还不清楚。针对这一问题，我们发现COPI复合体也启动了小管的形成，这参与了促进高尔基体内顺行运输和高尔基体带的形成。因此，在目前的更新中，我们建议确定预计在COPI小管与目标隔室对接过程中起作用的系链，并确定货物在这些小管中的运输方式。其次，我们注意到使ARF1失活的GTP酶激活蛋白(GAP)，ARFGAP1，以前已经被发现具有一个新的作用，也作为外壳成分。然而，关于这一作用一直存在争议，特别是在使用脂质体而不是高尔基膜的COPI重建研究中。因此，我们建议解释为什么脂质体为基础的系统不能重建ARFGAP1的外壳功能。此外，我们将澄清这一差距对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控COPI囊泡形成的新机制，并建议进一步剖析这一调控是如何实现的。第四，我们最近还阐明了一种关键的脂类--磷脂酸(PA)在COPI囊泡分裂中发挥作用的复杂性。因此，我们还将寻求对巴勒斯坦权力机构如何在这一过程中发挥作用的更好的机械性理解。总之，这些拟议的研究可能不仅会促进对COPI运输的机械性理解，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，我们的结果也可能为多种人类疾病的发生提供新的分子见解。 与公共健康相关：我们研究蛋白质和膜是如何在细胞内运输的。我们预计，我们提议的研究将促进对这种运输的基本理解。此外，由于这一过程对细胞功能的多个方面至关重要，我们进一步期待将关键的分子洞察力传递到导致人类疾病的病理事件中。

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.