Metal Homeostatic Mechanisms in Yeast

描述（由申请人提供）：本提案讨论了酿酒酵母作为真核生物模型的新型线粒体-细胞质信号通路。在第一个线粒体-细胞质途径中，我们发现了一种不同于金属伴侣铜穿梭的铜运输新范式。该途径涉及线粒体内用于细胞色素c氧化酶（CcO）和超氧化物歧化酶-1 （Sod1）金属化的铜配体（CuL）复合物的候选易位。除酵母外，CuL复合物是人成纤维细胞、小鼠肝脏和拟南芥线粒体铜的重要来源。我们假设Cu(I)与细胞质中丰富的自由配体结合后，CuL复合体通过一个尚未确定的线粒体转运体转运到线粒体。我们的目标是阐明CuL复合物的结构，验证其在线粒体基质中的易位，并确定转运蛋白为三个主要目标。这些目标的研究将涉及分析和结构生物学方法来推断配体结构，并结合细胞和体外线粒体进口研究来表征易位途径。我们将尝试通过基因筛选和评估呼吸缺陷突变体库来识别转运蛋白。第二种线粒体-细胞质途径与Aft1转录激活子的铁感应有关。在富含铁的细胞中，Aft1的转录激活被线粒体输出蛋白Atm1发出的信号所抑制。Aft1的功能与线粒体铁状态相耦合，因为许多酵母突变体表现出线粒体铁积累，表现出组成性的Aft1活性。我们的目标是确定在这些突变体中Aft1的铁抑制被阻断的机制。Aft1对铁的感知依赖于两种glutaredoxins和两种新发现的蛋白Fra1和Fra2。我们的目的是阐明这些蛋白如何介导铁对Aft1的抑制。人类细胞中有Atm1和Grx3/4的同源物，Atm1的人类同源物ABCB7在铁稳态中具有明确的作用。我们最近的研究表明存在可膨胀的基体铁池。这个铁池在铁-硫簇生物发生受损的酵母突变体中扩大，并可能导致超氧化物歧化酶-2 （so2）的错金属化和铁响应转录激活因子Aft1的组成性激活。本基金的统一主题是在基质中存在生物可利用的铜和铁池，以及线粒体-细胞质串扰是包括人类在内的真核生物中铜和铁稳态的关键组成部分。线粒体和细胞核之间的串扰在许多代谢疾病和紊乱中具有广泛的意义。公共卫生相关性：线粒体内铜配体复合物对细胞色素氧化酶和超氧化物歧化酶-1的铜金属化与人体生理学有关。细胞色素氧化酶组装缺陷导致新生儿心肌病患者出现呼吸窘迫。线粒体超氧化物歧化酶-1的突变与家族性肌萎缩性侧索硬化症的病因有关。已知人类ABCB7线粒体铁输出蛋白功能障碍可导致x连锁铁母细胞贫血伴共济失调。

DESCRIPTION (provided by applicant): This proposal addresses novel mitochondrial-cytoplasm signaling pathways in Saccharomyces cerevisiae as a model eukaryote. In the first mitochondrial-cytoplasm pathway, we discovered a new paradigm in copper trafficking distinct from metallochaperone copper shuttling. This pathway involves the candidate translocation of a copper-ligand (CuL) complex used in metallation of cytochrome c oxidase (CcO) and superoxide dismutase-1 (Sod1) within the mitochondria. In addition to yeast, the CuL complex is a significant source of mitochondrial copper in human fibroblasts, mouse liver and Arabidopsis. We postulate that translocation of the CuL complex into the mitochondria occurs through a yet unidentified mitochondrial transporter after Cu(I) is bound to an abundant free ligand in the cytoplasm. We aim to elucidate the structure of the CuL complex, verify its translocation into the mitochondrial matrix and identify the transporters as three major objectives. Studies in these objectives will involve analytical and structural biology approaches to deduce the ligand structure and a combination of cellular and in vitro mitochondrial import studies to characterize the translocation pathway. We will attempt to identify the transporters through a genetic screen and the evaluation of a bank of respiratory deficient mutants. A second mitochondrial-cytoplasm pathway relates to iron sensing by the Aft1 transcriptional activator. Transcriptional activation by Aft1 is inhibited in iron-replete cells by a signal emanating from the mitochondrial exporter Atm1. Aft1 function is coupled to the mitochondrial iron status, as a number of yeast mutants exhibiting mitochondrial iron accumulation show constitutively active Aft1. We aim to determine the mechanism by which iron-inhibition of Aft1 is blocked in these mutants. Iron sensing by Aft1 is dependent on two glutaredoxins and two newly identified proteins Fra1 and Fra2. We aim to elucidate how these proteins mediate Fe-inhibition of Aft1. Human cells have homologs to Atm1 and Grx3/4 with the human ortholog of Atm1, ABCB7, having a clear role in iron homeostasis. Our recent studies suggest that an expandable matrix iron pool exists. This iron pool is expanded in yeast mutants impaired in iron-sulfur cluster biogenesis and can lead to mis-metallation of superoxide dismutase-2 (Sod2) and constitutive activation of the iron-responsive transcriptional activator Aft1. The unifying themes in this grant are the presence of bioavailable pools of copper and iron within the matrix and mitochondrial-cytoplasm cross talk as a key component in copper and iron homeostasis in eukaryotes including humans. Cross talk between mitochondria and the nucleus has wide implications in a number of metabolic diseases and disorders. PUBLIC HEALTH RELEVANCE: Copper metallation of cytochrome oxidase and superoxide dismutase-1 by the copper- ligand complex within the mitochondria is relevant in human physiology. Deficiencies in cytochrome oxidase assembly lead to respiratory distress in humans presenting as neonatal cardiomyopathies. Mutants of the mitochondrial superoxide dismutase-1 are implicated in the etiology of familial amyotrophic lateral sclerosis. Dysfunction in the human ABCB7 mitochondrial iron exporter is known to cause X-linked sideroblastic anemia with ataxia.