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.

项目总结/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 内溶酶体系统的那些需要正确的脂质和蛋白质组成来完成它们的作用。一 囊泡运输的一个重要方面是脂质种类在外表面的不对称组织， 细胞膜的细胞面小叶。这种膜的不对称性已被证明是关键的蛋白质 运输、信号转导和凋亡。膜的不对称性是由IV型P型ATP酶建立的 （P4-ATP酶），其利用ATP水解将脂质底物从管腔或细胞外小叶转运至 细胞膜的细胞面小叶。P4-ATP酶缺陷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。为了正常的P4-ATP酶功能， 正确定位到适当的膜是至关重要的。酵母菌中的五种P4-ATP酶 已知Dnf 1、Dnf 2、Neo 1和Drs 2定位于质膜（PM）和/或高尔基体， 作为贩运路线的一部分，通过内吞途径旅行。在这项研究中，我试图确定 P4-ATP酶的循环和逆行运输途径，以及这些翻转酶如何与 这些途径的组成部分。核内体和核外体之间四种主要运输途径的关键组分 删除高尔基体（包括Drs 2/Rcy 1/COPI、Snx 4、retromer和AP-1/Clatherin）以确定途径 Dnf 1/Dnf 2 PM定位和Neo 1/Drs 2高尔基体定位所需的。删除retromer组分 包括Vps 35、Vps 5、Vps 17和Snx 3在内的多个基因的表达导致了Dnf 1、Dnf 2、Neo 1和Drs 2在液泡中的错误定位。 这些数据表明，逆转录酶在P4-ATP酶的正确定位中发挥主要作用，尽管逆转录酶在P4-ATP酶的正确定位中发挥次要作用。 检测到Rcy 1和Snx 4。此外，初步数据表明，有一种新的retromer识别 Dnf 1和Dnf 2的C-末端尾内的基序。这些结果表明，逆转录酶的损失导致显著的 由于P4-ATP酶向液泡的丢失，因此应该引起膜组织的重大变化。 Dnf 1/Dnf 2和retromer的人类直系同源物中的缺陷与内体功能障碍有关 导致帕金森病这项研究可以帮助阐明为什么这些蛋白质的突变会导致 疾病