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.

我们已经使用了全基因组的方法，以确定基因参与的反应，铁剥夺和铁超载的芽殖酵母，酿酒酵母。酵母通过激活铁吸收系统、动员储存的铁和转移到铁非依赖性代谢途径来响应铁剥夺。一种类型的铁摄取系统在铁剥夺过程中激活，特异性运输铁-铁载体螯合物，如铁色素（FC）。Arn 1是酿酒酵母中的一种FC转运蛋白，其胞内转运部分受FC与转运蛋白结合的控制。在不存在FC的情况下，Arn 1直接从高尔基体分选到内体。FC结合触发了Arn 1向质膜的再分布，而FC转运与Arn 1在质膜和内体之间的循环有关。我们确定，网格蛋白接头Gga 2和泛素化的Rsp 5泛素连接酶所需的运输的Arn 1。Gga 2是高尔基体将Arn 1运输到内体所必需的，Arn 1从内体被分选到液泡进行降解。虽然Gga 2被报道作为泛素化货物的受体，但Gga 2不需要泛素残基的结合来介导Arn 1的运输。进入液泡腔的运输依赖于Rsp 5的泛素化和Gga 2结合泛素的能力。质膜积累也不需要通过retromer复合物或Snx 4的逆行运输。如果没有这种泛素化，Arn 1仍然停留在质膜上，在那里它是活跃的运输。Arn 1的氨基末端的赖氨酸残基簇上的多聚泛素链优先修饰的transporter.Trafficking的Arn 1从TGN到液泡的腔需要网格蛋白衔接蛋白Gga 2的活性，它识别货物蛋白在TGN和分类到网格蛋白包被的囊泡内体和液泡的目的。虽然Gga 2已被表征为泛素受体，我们已经表明，Gga 2的泛素结合是不需要的TGN的内体运输的Arn 1，但需要为随后的排序的Arn 1到腔的空泡。网格蛋白衔接子Ent 3 p和Ent 4p也参与Arn 1 p的TGN到液泡分选，并且Arn 1的氨基末端中的短肽是这些相互作用所需的。 为了确定Arn 1运输的全球细胞需求，并确定参与蛋白质运输的新的细胞组分，我们设计了一个屏幕，以确定正确的Arn 1运输所需的所有开放阅读框架。与NCI的Munira Basrai合作，我们已经将GFP标记的Arn 1版本工程化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。