Cell Biology of Iron Transport

铁转运的细胞生物学

• 批准号: 7967559
• 负责人: Caroline Philpott
• 金额: $ 30.13万
• 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/7967559
• 关键词: Active Biological Transport; Adaptor Signaling Protein; Ataxia; Binding; Biochemical; Biological; Cell membrane; Cells; Cellular biology; Clathrin Adaptors; Clathrin-Coated Vesicles; Collaborations; Collection; Complex; Copper; Defect; Endosomes; Engineering; Eukaryota; Ferrichrome; Genes; Genetic; Genome; Golgi Apparatus; Hereditary Disease; Hereditary hemochromatosis; Homeostasis; Human; Intracellular Transport; Iron; Iron Overload; Lysine; Mediating; Metabolic Pathway; Metabolism; Nutrient; Open Reading Frames; Organism; Peptides; Polyubiquitin; Process; Proteins; Regulation; Reporting; Role; Saccharomyces cerevisiae; Saccharomycetales; Siderophores; Sorting - Cell Movement; System; Toxin; Ubiquitin; Ubiquitination; Vacuole; Yeasts; Zinc; deprivation; design; human disease; insight; 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.Trafficking of Arn1 from the TGN to the lumen of the vacuole requires the activity of the clathrin-adaptor protein Gga2, which recognizes cargo proteins at the TGN and sorts them into clathrin-coated vesicles destined for the endosome and vacuole. Although Gga2 has been characterized as a ubiquitin receptor, we have shown that ubiquitin binding by Gga2 is not required for the TGN-to-endosome trafficking of Arn1, but is required for the subsequent sorting of Arn1 into the lumen of the vacuole. The clathrin adaptors Ent3p and Ent4p are also involved in TGN-to-vacuole sorting of Arn1p, and short peptides in the amino terminus of Arn1 are required for these interactions. 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.

我们使用了全基因组方法来鉴定出芽酵母（Saccharomyces cerevisiae）中参与铁剥夺和铁过载反应的基因。酵母通过激活铁摄取系统、调动储存的铁以及转向铁独立的代谢途径来应对铁的缺乏。在缺铁过程中，一种类型的铁摄取系统被激活，特异地运输铁铁载体螯合物，如铁铬铁（FC）。酿酒酵母菌中FC转运体Arn1的细胞内转运部分受FC与转运体结合的控制。在没有FC的情况下，Arn1直接从高尔基体分选到核内体。FC结合触发Arn1向质膜的再分配，而FC转运则与Arn1在质膜和核内体之间的循环有关。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。高尔基体内转运Arn1需要Gga2， Arn1从内体中被分类到液泡中进行降解。尽管据报道Gga2作为泛素化货物的受体，但Gga2介导Arn1运输并不需要与泛素残基结合。空泡腔的转运依赖于Rsp5的泛素化和Gga2结合泛素的能力。通过反转录复合体或Snx4的逆行转运也不需要质膜积累。没有这种泛素化，Arn1留在质膜上，在那里它活跃于运输。Arn1被转运体氨基端赖氨酸残基簇上的多泛素链优先修饰。将Arn1从TGN运输到液泡的管腔需要网格蛋白衔接蛋白Gga2的活性，该蛋白识别TGN上的货物蛋白并将其分类到网格蛋白包被的囊泡中，最终到达核内体和液泡。虽然Gga2被认为是一种泛素受体，但我们已经证明，Gga2与泛素的结合并不是将Arn1转运到tgn -核内体所必需的，而是随后将Arn1分选到液泡管腔所必需的。网格蛋白接头Ent3p和Ent4p也参与了Arn1的tgn到液泡的分选，Arn1的氨基端需要短肽来进行这些相互作用。