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: 8413049
• 负责人: J Christopher Fromme
• 金额: $ 28.33万
• 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/8413049
• 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; 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; Life; Lipids; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Modeling; Molecular; Morphology; Mutagenesis; Mutation; N-terminal; Neurons; Nucleotides; Organelles; Phosphatidylinositol Phosphates; Proteins; Recruitment Activity; Regulation; Relative (related person); Research; Role; Sorting - Cell Movement; Surface; Testing; Yeasts; cell growth; cellular imaging; fluorescence imaging; human disease; in vitro Assay; in vivo; insight; 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）：许多人类疾病都是由亚细胞膜和蛋白质分选机制及其调节器的缺陷引起的。高尔基复合体是真核细胞中的主要分选细胞器，Arf GTPase 家族的蛋白质几乎控制着高尔基复合体的所有运输。 Arf GTP 酶在反高尔基体网络中被人类中的 Arf-GEF（鸟嘌呤核苷酸交换因子）蛋白 BIG1/ARFGEF1 和 BIG2/ARFGEF2 以及酵母中的同源 Sec7p 蛋白激活。尽管 Arf 激活的基本机制（GEF 结构域进行的核苷酸交换）已得到很好的表征，但 Arf-GEF 功能如何在高尔基体膜上进行调节尚不清楚。我们的实验室最近发现Sec7p表现出两种不同的自动调节行为：自动抑制和正反馈激活；这些行为取决于 Sec7p 的 N 末端和 C 末端区域，包括一个新的膜募集结构域。酵母 Sec7p 蛋白与其人类同源物之间的高度序列保守性表明这些调节行为很可能与人类细胞相关。该项目的长期目标是了解高尔基体定位的 Arf-GEF 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 Sec7p 自抑制机制和N-末端。我们将使用体外测定和诱变来剖析自抑制和 N 末端功能的分子机制。 2）表征正反馈的分子机制。体外测定将用于表征 Arf1p-GTP 和 Sec7p 膜募集结构域之间的相互作用，并将这种相互作用的亲和力与 Arf1p-GTP 对其效应子的亲和力进行比较。 3)确定Sec7p自动调节在体内蛋白质和膜运输中的作用。将使用电子和荧光显微镜、经典生化运输测定和活细胞成像来测试 Sec7p 自动调节突变体对细胞生长、高尔基体形态、Sec7p 定位和货物分选的体内影响。 4）测试高尔基体Arf-GEF自动调节的普遍性。我们将使用体外测定来确定 BIG1、BIG2 和 GBF1（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。拟议的研究对于我们了解高尔基体的膜和蛋白质运输至关重要，因为 Arf-GEF 是该细胞器中运输载体形成的主要调节者。我们希望我们的研究能够建立一个清晰的模型，说明高尔基体中 Arf GTPases 的激活如何在时空上受到调节。