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/文摘