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.

我们使用全基因组方法来鉴定参与芽殖酵母（酿酒酵母）铁缺乏和铁过载反应的基因。酵母通过激活铁吸收系统、动员储存的铁以及转向不依赖铁的代谢途径来应对铁缺乏。缺铁期间激活的一种铁吸收系统专门转运铁铁载体螯合物，例如铁铬 (FC)。 Arn1（酿酒酵母中的 FC 转运蛋白）的细胞内运输部分受到 FC 与转运蛋白结合的控制。在没有 FC 的情况下，Arn1 直接从高尔基体分选到内体。 FC 结合触发 Arn1 重新分布到质膜，而 FC 运输则与 Arn1 在质膜和内体之间的循环相关。我们确定网格蛋白接头 Gga2 和 Rsp5 泛素连接酶的泛素化是 Arn1 运输所必需的。 Gga2 是高尔基体向内体运输 Arn1 所必需的，Arn1 从内体分选到液泡进行降解。尽管据报道 Gga2 可以作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。进入液泡腔取决于 Rsp5 的泛素化以及 Gga2 结合泛素的能力。通过逆转录体复合物或 Snx4 的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 仍保留在质膜上，在那里它具有活跃的运输功能。 Arn1 优先在转运蛋白氨基末端的赖氨酸残基簇上用多聚泛素链进行修饰。 为了确定 Arn1 运输的全球细胞要求并识别参与蛋白质运输的新细胞成分，我们设计了一个屏幕来识别正确 Arn1 运输所需的所有开放阅读框。我们与 NCI 的 Munira Basrai 合作，将 GFP 标记的 Arn1 版本设计到酵母缺失突变体集合中，并使用该集合来识别具有蛋白质运输缺陷的缺失突变体。