Regulation of Arf GTPase activation at the trans-Golgi network

描述(申请人提供)：许多人类疾病是由亚细胞膜和蛋白质分选机械及其调节器的缺陷引起的。高尔基复合体是真核细胞的主要分选细胞器，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酶在人的反式高尔基体网络上被鸟嘌呤核苷酸交换因子(GNP)蛋白BIG1/ARFGEF1和BIG2/ARFGEF2激活，在酵母中被同源的Sec7p蛋白激活。虽然Arf激活的基本机制--通过全球环境基金结构域进行核苷酸交换--已经被很好地描述，但Arf-全球环境基金功能是如何在高尔基膜上调节的还不清楚。我们实验室最近发现，Sec7p表现出两种不同的自我调节行为：自我抑制和正反馈激活；这些行为取决于Sec7p的N端和C端的区域，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，这些调控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特定目标来获得：1)表征Sec7P自身抑制机制和N末端的作用。我们将使用体外试验和诱变来剖析自身抑制和N末端功能的分子机制。2)研究正反馈的分子机制。体外实验将用来表征Arf1p-GTP与Sec7p的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对其效应器的亲和力进行比较。3)确定Sec7p自身调节在体内蛋白质和膜转运中的作用。使用电子显微镜和荧光显微镜、经典的生化转运分析和活细胞成像，将测试Sec7p自动调节突变体对细胞生长、高尔基形态、Sec7p定位和货物分类的体内影响。4)检验高尔基体自动调节的通用性。我们将使用体外实验来确定BIG1、BIG2和GBF1(人类早期高尔基体定位的Arf-Global)是否也表现出自我调节行为。这项拟议的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-GEF是该细胞器运输载体形成的主要调节者。我们希望我们的研究将导致一个明确的模型，如何在高尔基体激活Arf GTP酶的时空调控。

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.