Mechanistic Studies of Iron Regulation in Yeast

描述（由申请人提供）：本提案的目的是揭示模式真核生物S.酿酒。为了维持最佳的细胞内铁水平，铁的转运和储存在从酵母到人类的所有真核细胞中受到严格调控。然而，我们在细胞和分子水平上对铁调节机制的理解存在重大差距。我们将通过梳理出酵母中铁调节的分子细节并定义铁信号通路中每个组分的作用来解决这些差距。在酵母中，单硫醇谷氧还蛋白Grx 3和Grx 4、BolA样蛋白Fra 2和氨肽酶P样蛋白Fra 1在控制铁响应性转录因子Aft 1的核质穿梭的铁响应性信号传导途径中一起起作用。在铁充足的条件下，该途径诱导Aft 1（以及可能是其副产物Aft 2）的二聚化，有利于它们定位于胞质溶胶。 我们已经证明Fra 2与Grx 3或Grx 4形成[2Fe-2S]2+桥连的异二聚体，并表征了这些配合物的Fe-S配位化学。此外，我们有强有力的证据表明，[2Fe-2S] Fra 2-Grx 3转移[2Fe-2S]簇到Aft 2，促进Aft 2二聚化。Aft 1/2二聚化，反过来，提出抑制铁调节子的激活。尽管我们在定义该信号通路中几个组分之间的分子相互作用方面取得了重大进展，但铁传感和调节机制的一些关键方面仍未得到解决，并将在本提案中得到解决。我们将揭示Fe-S从Fra 2-Grx 3/4转移到Aft 1和Aft 2的机制细节，并通过使用诱变，生化分析，和生物物理光谱，以检查簇转移到Aft 1和Aft 2的动力学和效率，并鉴定Grx 3/Grx 4/Fra 1/Fra 2/Aft 1/Aft 2中对供体-靶识别和Fe-2的关键残基。S转移（目标1）。我们将测试Fra-Grx复合物是否将Fe-S簇转移到Aft 1/2并通过确定Fra 2、Grx 3/4或Fra 1中的突变如何影响铁信号传导途径内的蛋白质-蛋白质相互作用、Fe与Aft 1/2的结合以及Aft 1/2的亚细胞定位和体内二聚化来诱导体内二聚化（Aim 2）。最后，我们将阐明Fra-Grx介导的Aft 1/2二聚化抑制铁调节子激活的机制，方法是使用体内和体外蛋白质-蛋白质和蛋白质-DNA相互作用试验（Aim 3）检测Fra-Grx介导的Aft 1/2二聚化是否破坏Aft 1/2向细胞核的移动、Aft 1/2与其DNA靶点的结合或转录共激活因子的募集。由于这一途径中的几个关键蛋白在人类中是保守的，并且对生存力至关重要，因此利用酵母系统来定义它们的功能和物理相互作用将提供对它们在人类铁代谢中的作用的基本理解。

DESCRIPTION (provided by applicant): This goal of this proposal is to uncover the molecular mechanisms for sensing and regulating intracellular iron in the model eukaryote S. cerevisiae. To maintain optimal intracellular iron levels, iron transport and storage is tightly regulated in al eukaryotic cells ranging from yeast to humans. However, there are significant gaps in our understanding of iron regulation mechanisms at the cellular and molecular level. We will address these gaps by teasing out the molecular details of iron regulation in yeast and defining the roles of each component in the iron signaling pathway. In yeast, the monothiol glutaredoxins Grx3 and Grx4, the BolA- like protein Fra2, and the aminopeptidase P-like protein Fra1 function together in an iron-responsive signaling pathway that controls nucleocytoplasmic shuttling of the iron-responsive transcription factor Aft1. Under iron replete conditions, this pathway induces dimerization of Aft1 (and presumably its paralog Aft2), favoring their localization to the cytosol. We have demonstrated that Fra2 forms [2Fe-2S]2+-bridged heterodimers with Grx3 or Grx4 and characterized the Fe-S coordination chemistry of these complexes. In addition, we have strong evidence that [2Fe-2S] Fra2-Grx3 transfers a [2Fe-2S] cluster to Aft2, facilitating Aft2 dimerization. Aft1/2 dimerization, in turn, is proposed to inhibit activation of the iron regulon. Despite our significant progress in defining the molecular interactions between several components in this signaling pathway, some key aspects of the iron sensing and regulation mechanism remain unresolved and will be addressed in this proposal. We will uncover the mechanistic details of Fe-S transfer from Fra2-Grx3/4 to Aft1 and Aft2 and determine the impact of Fra1 on this process by using mutagenesis, biochemical analysis, and biophysical spectroscopy to examine the kinetics and efficiency of cluster transfer to Aft1 and Aft2 and identify residues in Grx3/Grx4/Fra1/Fra2/Aft1/Aft2 that are critical for both donor-target recognition and Fe-S transfer (Aim 1). We will test whether the Fra-Grx complex transfers an Fe-S cluster to Aft1/2 and induces dimerization in vivo by determining how mutations in Fra2, Grx3/4, or Fra1 affect protein-protein interactions within the iron signaling pathway, Fe binding to Aft1/2, and Aft1/2 subcellular localization and dimerization in vivo (Aim 2). Finally, we will elucidate the mechanism by which Fra-Grx mediated Aft1/2 dimerization inhibits activation of the iron regulon by testing if Fra-Grx-mediated dimerization of Aft1/2 disrupts movement of Aft1/2 to the nucleus, binding of Aft1/2 to its DNA targets, or recruitment of transcriptional co-activators using both in vivo and in vitro protein-protein and protein-DNA interaction assays (Aim 3). Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism.