Cell Biology of Iron Transport

铁转运的细胞生物学

• 批准号: 7734201
• 负责人: Caroline Philpott
• 金额: $ 36.54万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734201
• 关键词: Active Biological Transport; Ataxia; Binding; Biochemical Genetics; Biological; Cell membrane; Cells; Cellular biology; Clathrin Adaptors; Collaborations; Collection; Complex; Copper; Defect; Endosomes; Engineering; Eukaryota; Eukaryotic Cell; Ferrichrome; Genes; Genetic; Genome; Golgi Apparatus; Hereditary Disease; Hereditary hemochromatosis; Homeostasis; Human; Intracellular Transport; Iron; Iron Overload; Lysine; Mediating; Metabolic Pathway; Metabolism; Numbers; Nutrient; Open Reading Frames; Organism; Polyubiquitin; Process; Regulation; Reporting; Role; Saccharomyces cerevisiae; Saccharomycetales; Siderophores; Sorting - Cell Movement; System; Toxin; Ubiquitin; Ubiquitination; Vacuole; Yeasts; Zinc; deprivation; design; endosome membrane; human disease; insight; intracellular protein transport; iron metabolism; metal metabolism; microorganism; mutant; protein transport; receptor; response; trafficking; ubiquitin ligase; uptake

We have used a whole-genome approach to identify genes that are involved in the response to iron deprivation and iron overload in the budding yeast, Saccharomyces cerevisiae. Yeast respond to iron deprivation by activating systems of iron uptake, by mobilizing stored iron, and by shifting to iron-independent metabolic pathways. One type of iron uptake system activated during iron deprivation specifically transports iron-siderophore chelates, such as ferrichrome (FC). The intracellular trafficking of Arn1, a FC transporter in Saccharomyces cerevisiae, is controlled in part by the binding of FC to the transporter. In the absence of FC, Arn1 is sorted directly from the Golgi to endosomes. FC binding triggers the redistribution of Arn1 to the plasma membrane, while FC transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. We determined that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi to endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Although Gga2 is reported to act as a receptor for ubiquitinated cargo, the binding of ubiquitin residues was not required for Gga2 to mediate trafficking of Arn1. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, and on the capacity of Gga2 to bind ubiquitin. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with poly-ubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter. In order to determine the global cellular requirements for Arn1 trafficking and to identify new cellular components involved in protein trafficking, we have designed a screen to identify all of the open reading frames necessary for proper Arn1 trafficking. In collaboration with Munira Basrai of NCI, we have engineered a GFP-tagged version of Arn1 into the yeast deletion mutant collection, and are using this collection to identify deletion mutants with defects in protein trafficking.

我们已经使用全基因组方法来确定在萌芽酵母中参与铁缺乏和铁超载反应的基因。酵母对缺铁的反应是通过激活铁摄取系统，动员储存的铁，并转移到铁不依赖的代谢途径。在缺铁期间激活的一种铁摄取系统专门运输铁-铁载体螯合物，如铁铬(Fc)。酿酒酵母中Fc转运蛋白Arn1的胞内转运部分受Fc与转运蛋白结合的控制。在没有Fc的情况下，Arn1直接从高尔基体到内体。Fc结合触发Arn1重新分布到质膜，而Fc转运与Arn1在质膜和内体之间的循环有关。我们确定Arn1的运输需要网状蛋白适配器GGa2和Rsp5泛素连接酶的泛素化。高尔基体需要GGa2来运输Arn1，Arn1是从内体分离到液泡中降解的。尽管GGa2被报道为泛素化货物的受体，但GGa2介导Arn1的运输并不需要泛素残基的结合。转运到空泡腔依赖于RSP5的泛素化，以及GGa2结合泛素的能力。通过逆转录复合体或Snx4逆行运输也不是质膜积累所必需的。如果没有这种泛素化，Arn1仍然留在质膜上，在那里它是活跃的运输。Arn1在转运蛋白氨基末端的一簇赖氨酸残基上优先被多泛素链修饰。 为了确定Arn1转运的全球细胞需求，并确定参与蛋白质转运的新的细胞成分，我们设计了一个屏幕来识别正确的Arn1转运所需的所有开放阅读框架。在与NCI的Munira Basrai的合作下，我们已经将GFP标记的Arn1版本设计到酵母缺失突变体库中，并正在使用该库来鉴定具有蛋白质运输缺陷的缺失突变体。