Functions and regulation of yeast Golgi Arf-like GTPases

描述（由申请人提供）：Ras相关GTP酶是多种信号通路的重要调节剂，包括细胞器生物发生和维持所需的信号通路。ARF和Rab亚家族的成员通过其下游效应子控制囊泡的生物发生和融合，并且它们被认为是高尔基体结构和功能的中心调节因子。我们一直在研究两个酵母ARF样GTP酶，Arl 1 p和ArI 3 p，这是高度保守的整个真核生物进化。Ar 1 p和ArI 3 p定位于高尔基体，在那里它们是蛋白质分选和通过高亲和力摄取途径对铁的高尔基体依赖性摄取所必需的。ArI 1 p和ArI 3 p的功能与Ypt 6p高尔基体Rab GT3的信号传导有关：Ypt 6p信号传导是ArI 3 p靶向和激活所必需的，ArI 3 p信号传导是ArI 1 p靶向和信号传导所必需的。本研究的目标是确定在这个GT3级联信号的链接因素，并阐明ArI 3 p和Arl 1 p调节高尔基体功能的机制。ArI 3 p是不寻常的，因为它不是豆蔻酰化的，但N-末端的甲基必须被乙酰化以靶向高尔基体。在具体目标1，我们将测试的假设，高尔基体膜蛋白，Sys 1 p，作为N-末端乙酰化Arl 3 p的高尔基体受体的功能。具体目标2中提出的实验将鉴定调节Arl 1 p和ArI 3 p的鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAP），以及分别将Ypt 6p和ArI 3 p的下游信号传导与ArI 3 p和Arl 1 p的激活联系起来的效应物。ARL GT突变体的高亲和力铁摄取系统的缺陷是由于未能向高尔基体的内腔提供铜离子，铜离子是已知的铁摄取辅因子。具体目标3中提出的实验将阐明Arl 1 p和ArI 3 p在用铜离子加载高尔基体中的作用。两种人类疾病，威尔逊病和门克病，是由于高尔基体定位的铜外排转运蛋白的无能，我们的研究将有助于了解这些蛋白质如何在细胞内定位和调节。

DESCRIPTION (provided by applicant): Ras-related GTPases are important regulators of a wide variety of signaling pathways, including those required for organelle biogenesis and maintenance. Through their downstream effectors, members of the ARF and Rab sub-families control vesicle biogenesis and fusion, and they are recognized as central regulators of Golgi structure and function. We have been investigating two yeast ARF-like GTPases, Arl1p and ArI3p, that are highly conserved throughout eukaryotic evolution. Ar1p and ArI3p are localized to the Golgi apparatus where they are required for protein sorting and for Golgi-dependent uptake of iron via the high affinity uptake pathway. The functions of Arl1p and ArI3p are linked to signaling by the Ypt6p Golgi Rab GTPase: Ypt6p signaling is required for targeting and activation of ArI3p, and signaling by ArI3p is required for targeting and signaling by Arl1p. The goals of this research proposal are to identify factors that link signaling in this GTPase cascade and to elucidate the mechanisms by which ArI3p and Arl1p regulate Golgi function. ArI3p is unusual in that it is not myristoylated, but the N-terminal Methionine residue must be acetylated to be targeted to the Golgi. In Specific Aim 1, we will test the hypothesis that the Golgi integral membrane protein, Sys1p, functions as a Golgi receptor for N-terminal acetylated Arl3p. The experiments proposed in Specific Aim 2 will identify the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that regulate Arl1p and ArI3p, and the effectors that link downstream signaling of Ypt6p and ArI3p to activation of ArI3p and Arl1 p, respectively. The defect the high affinity iron uptake system of ARL GTPase mutants is due to a failure to provide copper ion, a known co-factor for iron uptake, to the lumen of the Golgi. The experiments proposed in Specific Aim 3 will elucidate the role of Arl1 p and ArI3p in loading the Golgi with copper ion. Two human disorders, Wilson's and Menke's diseases, result from an inability of Golgi-localized copper efflux transporters and our research will contribute to an understanding how these proteins are localized and regulated within the cell.