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 蛋白质和脂类在细胞内的运输对细胞功能和细胞器至关重要，包括 内溶酶体系统的那些，需要正确的脂肪和蛋白质组成来完成它们的作用。一 囊泡运输的重要方面是脂类跨表面和 细胞膜的细胞面小叶。这种膜的不对称性已被证明是蛋白质的关键 运输、信号转导和细胞凋亡。膜的不对称性是由IVP型ATPase建立的 (P4-ATPase)，它利用ATP水解将脂质底物从管腔或细胞外的小叶运输到 细胞膜的细胞面小叶。P4-ATPase缺陷扰乱高尔基体小泡介导的蛋白质运输 和内小体膜，并引起内小体和溶酶体的超酸化。我们的目标是 了解P4-ATPase在囊泡介导的蛋白质运输中的作用。对于正确的P4-ATPase功能， 正确定位到合适的膜是至关重要的。酵母菌中的五种P4-ATPase 已知Cerevisiae、Dnf1、Dnf2、Neo1和Drs2定位于质膜(PM)和/或高尔基体和 作为它们贩运路线的一部分，它们通过内吞途径旅行。在这项研究中，我试图确定 P4-ATPase的回收和逆行运输途径，以及这些Flippase如何与 这些路径的组成部分。四个主要运输途径的关键组成部分在内体和 这些高尔基体包括Drs2/Rcy1/CoPI，Snx4，Retromer和AP-1/clathrin，以确定路线 Dnf1/Dnf2 PM本地化和Neo1/Drs2高尔基本地化需要。逆转录病毒组分的缺失 包括Vps35、Vps5、Vps17和Snx3，导致Dnf1、Dnf2、Neo1和Drs2错误定位于液泡。 这些数据表明，逆转聚体在P4-ATPase的正确定位中起主要作用，尽管在P4-ATPase的定位中作用不大 检测到Rcy1和Snx4基因。此外，初步数据表明，存在一种新的逆转录识别 Dnf1和Dnf2的C-末端尾部的基序。这些结果表明，逆转录聚体的丢失会导致大量的 P4-ATPase丢失到液泡中，因此应该会引起膜组织的重大变化。 人类Dnf1/Dnf2同源基因和逆转录增殖体的缺陷与内体功能障碍有关 导致帕金森氏症。这项研究可能有助于解释为什么这些蛋白质的突变会导致 疾病。