Determining the role of retromer and P4-ATPase interactions in cellular functions

确定逆转录酶和 P4-ATP 酶相互作用在细胞功能中的作用

• 批准号: 10677963
• 负责人: Mariana De Jesus Jimenez
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677963
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Alleles; Alzheimer' s Disease; Amino Acid Motifs; Apoptosis; Back; Binding Sites; Biological; C-terminal; Capsid Proteins; Carrier Proteins; Cell membrane; Cell physiology; Cells; Charge; Chimeric Proteins; Clathrin; Clathrin-Coated Vesicles; Coat Protein Complex I; Collection; Complex; Cytoplasm; Cytoplasmic Tail; Data; Disease; Endosomes; Excision; Family; Functional disorder; Glucosylceramides; Goals; Golgi Apparatus; Health; Human; Knock-out; Lecithin; Length; Life; Link; Lipids; Lysosomes; Mediating; Membrane; Metabolic; Minor; Modeling; Monitor; Morphology; Multivesicular Body; Mutation; N-terminal; Neurologic; Organelles; Orthologous Gene; Parkinson Disease; Pathway interactions; Pattern; Phosphatidylethanolamine; Phosphatidylserines; Phospholipids; Play; Point Mutation; Process; Property; Proteins; Recycling; Regulation; Role; Route; Saccharomyces cerevisiae; Signal Transduction; Sorting; Sphingolipids; Structure; System; Tail; Temperature; Testing; Transcription Factor AP-1; Travel; Vacuole; Vesicle; Yeasts; endosome membrane; extracellular; late endosome; lipid transport; nervous system disorder; novel; protein transport; sorting nexins; trafficking; trans-Golgi Network; vesicle transport

PROJECT SUMMARY/ABSTRACT The transport of proteins and lipids throughout the cell is crucial for cellular function and organelles, including those of the endolysosomal system, require the correct lipid and protein composition to complete their roles. One important aspect of vesicular trafficking is the asymmetric organization of lipid species across the exofacial and cytofacial leaflets of the membrane. This membrane asymmetry has been shown to be critical for protein trafficking, signal transduction and apoptosis. Membrane asymmetry is established by type-IV P-type ATPases (P4-ATPase), which utilize ATP hydrolysis to transport lipid substrates from the luminal or extracellular leaflet to the cytofacial leaflet of membranes. P4-ATPase deficiency disrupts vesicle-mediated protein transport from Golgi and endosomal membranes and causes hyperacidification of endosomes and lysosomes. Our goal is to understand the role of P4-ATPases in vesicle-mediated protein transport. For proper P4-ATPase function, correct localization to appropriate membranes is essential. Of the five P4-ATPases in Saccharomyces cerevisiae, Dnf1, Dnf2, Neo1 and Drs2 are known to localize to the plasma membrane (PM) and/or Golgi and travel through the endocytic pathway as part of their trafficking itineraries. In this study, I seek to determine the recycling and retrograde trafficking pathways traveled by P4-ATPases, and how these flippases interact with components of those pathways. Key components of four major trafficking pathways between endosomes and the Golgi including Drs2/Rcy1/COPI, Snx4, retromer and AP-1/Clathrin, were deleted to determine the routes required for Dnf1/Dnf2 PM localization and Neo1/Drs2 Golgi localization. Deletion of retromer components including Vps35, Vps5, Vps17, and Snx3 led to mislocalization of Dnf1, Dnf2, Neo1 and Drs2 to the vacuole. These data suggest a primary role for retromer in proper localization of P4-ATPases although a minor role for Rcy1 and Snx4 was detected. In addition, preliminary data suggest that there is a novel retromer recognition motif within the C-terminal tail of Dnf1 and Dnf2. These results indicate that loss of retromer leads to a substantial loss of P4-ATPases to the vacuole and should therefore cause major changes in membrane organization. Deficiencies in the human orthologs of Dnf1/Dnf2 and retromer have been linked to endosomal dysfunction leading to Parkinson's Disease. This study could help elucidate why mutations in these proteins cause the disease.

项目概要/摘要 蛋白质和脂质在整个细胞中的运输对于细胞功能和细胞器至关重要，包括 内溶酶体系统的细胞需要正确的脂质和蛋白质组成才能完成其作用。一 囊泡运输的一个重要方面是脂质种类在外面部和面部的不对称组织。 膜的细胞面小叶。这种膜不对称性已被证明对蛋白质至关重要 运输、信号转导和细胞凋亡。 IV 型 P 型 ATP 酶建立膜不对称性 (P4-ATPase)，利用 ATP 水解将脂质底物从管腔或细胞外小叶转运至 膜的细胞面小叶。 P4-ATP酶缺乏破坏囊泡介导的高尔基体蛋白质转运 和内体膜并引起内体和溶酶体的过度酸化。我们的目标是 了解 P4-ATP 酶在囊泡介导的蛋白质转运中的作用。为了保证 P4-ATP 酶的正常功能， 正确定位到适当的膜至关重要。酵母菌中的五种 P4-ATP 酶 已知酿酒酵母、Dnf1、Dnf2、Neo1 和 Drs2 定位于质膜 (PM) 和/或高尔基体， 作为其贩运路线的一部分，他们会通过内吞途径进行旅行。在这项研究中，我试图确定 P4-ATP酶经过的回收和逆行运输途径，以及这些翻转酶如何与 这些途径的组成部分。内体和内体之间四种主要运输途径的关键组成部分 删除高尔基体，包括 Drs2/Rcy1/COPI、Snx4、retromer 和 AP-1/Clathrin，以确定路径 Dnf1/Dnf2 PM 定位和 Neo1/Drs2 高尔基体定位所需。删除retromer组件 包括 Vps35、Vps5、Vps17 和 Snx3 导致 Dnf1、Dnf2、Neo1 和 Drs2 错误定位到液泡。 这些数据表明，retromer 在 P4-ATPase 的正确定位中起主要作用，尽管对于 检测到 Rcy1 和 Snx4。此外，初步数据表明存在一种新的逆转录酶识别 Dnf1 和 Dnf2 的 C 末端尾部内的基序。这些结果表明，逆转录酶的损失导致大量 P4-ATP酶损失到液泡中，因此会引起膜组织的重大变化。 人类 Dnf1/Dnf2 直系同源物和逆转录酶的缺陷与内体功能障碍有关 导致帕金森病。这项研究可能有助于阐明为什么这些蛋白质的突变会导致 疾病。