Cell Biology of Iron Transport

铁转运的细胞生物学

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

我们已经使用了一种全基因组方法来鉴定与酿酒酵母酵母中对铁剥夺和铁超载反应有关的基因。酵母通过激活铁吸收系统，动员储存的铁以及转移到铁独立的代谢途径来应对铁的剥夺。铁剥夺期间激活的一种铁摄取系统专门运输铁螺旋体螯合物，例如铁晶（FC）。 ARN1的细胞内运输是酿酒酵母中的FC转运蛋白，部分通过FC与转运蛋白的结合来控制。在没有FC的情况下，ARN1直接从高尔基体到内体排序。 FC结合触发了ARN1与质膜的重新分布，而FC转运与质膜和内体之间的ARN1循环有关。我们确定RSP5泛素连接酶的网旋蛋白适配器GGA2和泛素化是ARN1运输所必需的。高尔基（Golgi）需要GGA2进行ARN1的内体贩运，该内体贩运从内体到液泡进行降解。尽管据报道GGA2充当泛素化货物的受体，但GGA2介导ARN1的贩运并不需要泛素残基的结合。运输到液泡腔内取决于RSP5的泛素化，以及GGA2结合泛素的能力。质膜积累也不需要通过缩回络合物或SNX4进行逆行运输。如果没有这种泛素化，ARN1留在质膜上，它具有运输活性。在转运蛋白的氨基末端的一组赖氨酸残基上，用聚泛素链优先修饰ARN1。为了确定ARN1运输的全球细胞要求并确定涉及蛋白质运输的新细胞成分，我们设计了一个屏幕来识别适当的ARN1运输所需的所有开放阅读框架。通过与NCI的Munira Basrai合作，我们将GFP标记的ARN1版本设计为酵母删除突变体集合，并正在使用此系列来识别具有蛋白质运输缺陷的缺失突变体。