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.

项目摘要/摘要 整个细胞中蛋白质和脂质的转运对于细胞功能和细胞器至关重要，包括 内溶性系统的那些需要正确的脂质和蛋白质组成才能完成其角色。一 囊泡贩运的重要方面是脂质物种不对称的组织， 膜的胞叶小叶。该膜不对称性已被证明对蛋白质至关重要 运输，信号转导和凋亡。膜不对称是由IV型P型ATPase建立的 （P4-ATPase），该（P4-ATPase）利用ATP水解将脂质底物从腔或细胞外叶传递到 膜的胞叶小叶。 P4-ATPase缺乏破坏囊泡介导的蛋白质从高尔基体转运 内体膜并引起内体和溶酶体的过度鉴定。我们的目标是 了解P4-ATP酶在囊泡介导的蛋白转运中的作用。对于适当的P4-ATPase函数， 正确地定位适当的膜是必不可少的。在糖疗法中的五个P4-ATP酶中 众所周知，酿酒酵母，DNF1，DNF2，NEO1和DRS2本地化于质膜（PM）和/或Golgi，以及 作为其贩运行程的一部分，穿越内吞途径。在这项研究中，我试图确定 P4-ATPases传播的回收和逆行贩运途径，这些Flippases如何与 这些途径的组成部分。内体和内体之间四个主要贩运途径的关键组成部分 删除了包括DRS2/RCY1/COPI，SNX4，RETOMER和AP-1/网格蛋白的Golgi，以确定路由 DNF1/DNF2 PM定位和NEO1/DRS2高尔基体定位所需。删除缩回组件 包括VPS35，VPS5，VPS17和SNX3导致DNF1，DNF2，NEO1和DRS2的错误定位到液泡中。 这些数据表明，逆转在适当定位P4-ATPases中的主要作用，尽管对 检测到RCY1和SNX4。此外，初步数据表明有一种新颖的逆转录识别 在DNF1和DNF2的C末端尾部内的基序。这些结果表明，逆转录者的损失导致了很大的 P4-ATP酶输给液泡，因此应导致膜组织的重大变化。 DNF1/DNF2和逆转录器的人类直系同源物中的缺陷与内体功能障碍有关 导致帕金森氏病。这项研究可以帮助阐明为什么这些蛋白质中的突变导致 疾病。