Functions and regulation of yeast Golgi Arf-like GTPases

描述(由申请人提供)：RAS相关的GTP酶是多种信号通路的重要调节者，包括细胞器生物发生和维持所需的那些。ARF和RAB亚家族的成员通过其下游效应器控制囊泡的生物发生和融合，它们被认为是高尔基体结构和功能的中央调节因子。我们一直在研究两种酵母ARF样GTP酶，Arl1p和ArI3p，它们在真核进化过程中高度保守。Ar1p和ArI3p定位于高尔基体，在高尔基体中，它们是蛋白质分离和通过高亲和力摄取途径依赖高尔基体摄取铁所必需的。Arl1p和ArI3p的功能与Ypt6p Golgi Rab GTP酶的信号转导有关：Arl3p的靶向和激活需要Ypt6p信号，Arl1p的靶向和信号转导需要ArI3p的信号。本研究的目的是确定在这一GTP酶级联中连接信号的因素，并阐明ArI3p和Arl1p调节高尔基体功能的机制。ArI3p的不同寻常之处在于它不是肉豆蔻酰化的，但N-末端的蛋氨酸残基必须乙酰化才能靶向高尔基体。在特定目标1中，我们将检验高尔基体完整膜蛋白Sys1p作为N末端乙酰化Arl3p的高尔基体受体的假设。在特定目标2中提出的实验将确定调节Arl1p和ArI3p的鸟核苷酸交换因子(GEF)和GTP酶激活蛋白(GAP)，以及分别将Ypt6p和ArI3p的下游信号与ArI3p和ArL1p的激活联系起来的效应器。ARL GTP酶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管腔提供铜离子，铜离子是已知的铁吸收的辅助因子。在特定目标3中提出的实验将阐明ARL1p和ArI3p在负载铜离子的高尔基体中的作用。人类的两种疾病，威尔逊病和门克病，是由于高尔基体定位的铜外流转运蛋白不能引起的，我们的研究将有助于理解这些蛋白是如何在细胞内定位和调节的。

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.