Sec7 Domain Arf Exchange Factors In Membrane Traffic

真核细胞的一个显著特征是其内部的膜组织。内膜结构是高度动态的，其完整性和维持依赖于持续的膜运输和蛋白质运输。小分子GTP酶ARF(ADP核糖化因子)在蛋白质转运的多个步骤中起着重要作用。ARF的GDP/GTP循环导致膜的脂质和蛋白质组成发生变化，从而调节膜结构。ARF研究得最多的作用是将胞质包衣复合体募集到膜上，使膜变形，产生运输中间体，并将货物蛋白浓缩到这些运输载体上。一类新发现的ARF效应物是脂质修饰酶，如磷脂酰肌醇-4-磷酸-5-激酶。ARF依赖鸟嘌呤核苷酸交换因子(Global)来激活。凯瑟琳·L·杰克逊·S博士是第一个在酿酒酵母中发现ARF环境因子的人，命名为Gea1p(在ARF上进行鸟嘌呤核苷酸交换的意思)。到目前为止，已经鉴定了十几种ARF全球环境基金蛋白。它们都有一个大约200个氨基酸的区域(“sec7结构域”)，该区域与酿酒酵母Sec7p蛋白的一个结构域同源，参与分泌。在体外，只有Sec7结构域是催化ARF上GDP/GTP交换的必要条件和充分条件。我们正在研究酿酒酵母Gea1p、Gea2p和Sec7p中三个ARF GEF的sec7结构域以及哺乳动物细胞中的两个sec7同源物BIG1和BIG2的体内和体外功能。已经确定了Gea1/2p、Sec7p和BIG2的Sec7结构域的一些相互作用伙伴。我们正在研究这些相互作用的物理性质和它们的生理作用。第二个主要项目是确定sec7结构域蛋白中的膜结合决定因素。所有这些蛋白质都是外周与膜结合的，目前尚不清楚膜相互作用是通过与脂类直接结合还是通过蛋白质-蛋白质相互作用来介导的。有趣的是，Gea2p的一个直接结合伙伴是跨膜结构域蛋白，因此这种相互作用正在探索潜在的膜靶向作用。

A distinguishing feature of eukaryotic cells is their internal membrane organization. Internal membrane stuctures are highly dynamic and their integrity and maintenance are dependent upon continual membrane trafficking and protein transport. The small GTPase ARF (ADP-Ribosylation Factor) plays an essential role in multiple steps of protein transport. The GDP/GTP cycle of ARF results in changes in both lipid and protein composition of membranes, thus modulating membrane structure. The best-studied role of ARF is recruitment of cytosolic coat complexes onto membranes, which can deform membranes to produce transport intermediates and concentrate cargo proteins into these transport carriers. A newly-discovered class of ARF effectors is lipid modifying enzymes such as phosphatidylinositol-4-phosphate 5-kinase. ARF relies on a guanine nucleotide exchange factor (GEF) to become activated. Dr. Catherine L. Jackson?s laboratory was the first to identify an ARF GEF, named Gea1p (for Guanine-nucleotide Exchange on ARF) in S. cerevisiae. There are now a dozen ARF GEF proteins characterized to date. They all share a region of approximately 200 amino acids (the "Sec7 domain") that is homologous to a domain of the Sec7p protein of S. cerevisiae, involved in secretion. The Sec7 domain alone is necessary and sufficient to catalyze GDP/GTP exchange on ARF in vitro. We are investigating the in vivo and in vitro functions of the Sec7 domains of three ARF GEFs in S.cerevisiae, Gea1p, Gea2p, and Sec7p, and two Sec7 orthologues in mammalian cells, BIG1 and BIG2. A number of interacting partners of the Sec7 domains of Gea1/2p, Sec7p an BIG2 have been identified. We are investigating both the physical properties of these interactions and their physiological roles. A second major project is the identification of membrane-binding determinants in the Sec7 domain proteins. All of these proteins are peripherally associated with membranes, and it is not known whether membrane interaction is mediated by direct association with lipids or through protein-protein interaction. Interestingly, one direct binding partner of Gea2p is a transmembrane-domain protein, so this interaction is being explored for a potential membrane-targeting role.