Regulation of Arf GTPase activation at the trans-Golgi network

跨高尔基体网络 Arf GTPase 激活的调节

• 批准号: 9000156
• 负责人: J Christopher Fromme
• 金额: $ 35.48万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9000156
• 关键词: Affinity; Behavior; Binding; Biochemical; Biological Assay; C-terminal; Cell Survival; Cells; Cellular Membrane; Cellular biology; Defect; Dimerization; Disease; Electron Microscopy; Embryonic Development; Eukaryotic Cell; Exhibits; Family; Feedback; Fluorescence Microscopy; GBF1 gene; GTP Binding; Genes; Goals; Golgi Apparatus; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Homologous Gene; Human; In Vitro; Individual; Knowledge; Lead; Lipids; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Modeling; Molecular; Morphology; Mutagenesis; Mutation; N-terminal; Neurons; Nucleotides; Organelles; Phosphatidylinositol Phosphates; Proteins; Recruitment Activity; Regulation; Research; Role; Sorting - Cell Movement; Surface; Testing; Yeasts; cell growth; fluorescence imaging; human disease; in vitro Assay; in vivo; insight; live cell imaging; membrane activity; mutant; novel; research study; temperature sensitive mutant; tool; trafficking; trans-Golgi Network

DESCRIPTION (provided by applicant): Many human diseases arise from defects in sub-cellular membrane and protein sorting machinery and its regulators. The Golgi complex is the primary sorting organelle in eukaryotic cells, and proteins of the Arf GTPase family control virtually all traffic out of the Golgi complex. Arf GTPases are activated at the trans- Golgi network by the Arf-GEF (Guanine nucleotide Exchange Factor) proteins BIG1/ARFGEF1 and BIG2/ARFGEF2 in humans, and by the homologous Sec7p protein in yeast. Although the basic mechanism of Arf activation - nucleotide exchange by the GEF domain - is well characterized, how Arf-GEF function is regulated at Golgi membranes is not known. Our lab has recently discovered that Sec7p exhibits two distinct autoregulatory behaviors: autoinhibition and positive feedback activation; these behaviors depend upon regions of the N-terminus and C-terminus of Sec7p, including a novel membrane recruitment domain. The high degree of sequence conservation between the yeast Sec7p protein and its human homologs indicates that these regulatory behaviors are very likely to be relevant in human cells. The long-term goal of this project is to understand how the essential function of the Golgi-localized Arf-GEFs is regulated, and will be obtained by pursuing the following four Specific Aims: 1) Characterize the Sec7p autoinhibitory mechanism and the role of the N-terminus. We will use in vitro assays and mutagenesis to dissect the molecular mechanisms of autoinhibition and N-terminal function. 2) Characterize the molecular mechanism of positive feedback. In vitro assays will be used to characterize the interaction between Arf1p-GTP and the membrane recruitment domain of Sec7p, and to compare the affinity of this interaction to the affinity of Arf1p-GTP for its effectors. 3) Determine the role of Sec7p autoregulation in protein and membrane trafficking in vivo. Sec7p autoregulatory mutants will be tested for their in vivo effects on cell growth, Golgi morphology, Sec7p localization, and cargo sorting, using electron and fluorescence microscopy, classical biochemical trafficking assays, and live-cell imaging. 4) Test the generality of Golgi Arf-GEF autoregulation. We will use in vitro assays to determine if BIG1, BIG2, and GBF1 (the human early-Golgi-localized Arf-GEF) also exhibit autoregulatory behavior. The proposed research is critical to our understanding of membrane and protein traffic at the Golgi, as Arf-GEFs are master regulators of transport-carrier formation at this organelle. We expect that our research will lead to a clear model for how activation of Arf GTPases at the Golgi is regulated spatiotemporally.

描述（由申请人提供）：许多人类疾病源于亚细胞膜和蛋白质分选机制及其调节剂的缺陷。高尔基复合体是真核细胞中的主要分选细胞器，并且Arf GTdR家族的蛋白质控制几乎所有从高尔基复合体出来的运输。Arf GTP酶在人中被Arf-GEF（鸟嘌呤核苷酸交换因子）蛋白BIG 1/ARFGEF 1和BIG 2/ARFGEF 2以及酵母中被同源Sec 7 p蛋白在反式高尔基体网络处激活。虽然Arf激活的基本机制-通过GEF结构域进行核苷酸交换-已得到很好的表征，但Arf-GEF功能如何在高尔基体膜上调节尚不清楚。我们的实验室最近发现，Sec 7 p表现出两种不同的自调节行为：自抑制和正反馈激活;这些行为依赖于Sec 7 p的N-末端和C-末端区域，包括一个新的膜募集结构域。酵母Sec 7 p蛋白及其人类同源物之间的高度序列保守性表明这些调控行为很可能与人类细胞相关。本项目的长期目标是了解高尔基体定位的Arf-GEFs的基本功能是如何调节的，并将通过追求以下四个具体目标来实现：1）表征Sec 7 p自抑制机制和N-末端的作用。我们将使用体外试验和诱变来剖析自抑制和N-末端功能的分子机制。2)描述正反馈的分子机制。体外试验将用于表征Arf 1 p-GTP与Sec 7 p的膜募集结构域之间的相互作用，并比较该相互作用的亲和力与Arf 1 p-GTP对其效应物的亲和力。3)确定Sec 7 p自身调节在体内蛋白质和膜运输中的作用。Sec 7 p自动调节突变体将测试其在体内对细胞生长，高尔基体形态，Sec 7 p定位和货物分选的影响，使用电子和荧光显微镜，经典的生化运输试验，活细胞成像。4)检验高尔基体Arf-GEF自动调节的通用性。我们将使用体外试验来确定BIG 1，BIG 2和GBF 1（人类早期高尔基体定位的Arf-GEF）是否也表现出自动调节行为。这项研究对于我们理解高尔基体的膜和蛋白质运输至关重要，因为Arf-GEFs是这个细胞器中运输载体形成的主要调节剂。我们希望我们的研究将导致一个明确的模型，如何激活的Arf GTP酶在高尔基体的时空调节。