Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

基本信息

批准号：
8046666
负责人：
HELEN L YIN
金额：
$ 0.54万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8046666
关键词：
1-Phosphatidylinositol 4-Kinase Acyltransferase Adaptor Signaling Protein Address Alzheimer&apos s Disease Behavior Binding Biology Capsid Proteins Cells Clathrin Clathrin Adaptors Cysteine Data Dependence Destinations Detection Development Docking Early Endosome Endosomes Epitopes Figs - dietary Generations Goals Golgi Apparatus Golgi Targeting Grant Growth Factor Guanosine Triphosphate Phosphohydrolases Heterodimerization Hybrids Immunoprecipitation Integral Membrane Protein Isotopes Life Lipids Lysosomes Medial Golgi Membrane Membrane Microdomains Membrane Protein Traffic Membrane Proteins Metabolic Modeling Movement Multivesicular Body Neurodegenerative Disorders Organelles Pathway interactions Phenotype Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphotransferases Population Production Protein Isoforms Proteins Proteomics RNA Interference Recording of previous events Recruitment Activity Recycling Regulation Role Route Sirolimus Site Small Interfering RNA Sorting - Cell Movement Testing Transferase Transmembrane Transport Vesicle adapter protein base cellular imaging density design human PHEMX protein inorganic phosphate late endosome membrane activity mutant overexpression palmitoylation phosphatidylinositol 4-phosphate protein protein interaction public health relevance research study secretory protein segregation tool trafficking trans-Golgi Network

项目摘要

DESCRIPTION (provided by applicant): Our overall hypothesis is that PI4KII? (phosphatidylinositol 4 kinase II?), which produces more than 50% of the phosphatidylinositol 4 phosphate (PI4P) in the Golgi, regulates Golgi functions through its localized production of PI4P. The PI4P itself establishes the Golgi's unique organelle identity by recruiting clathrin adapter proteins, such as AP-1, through the coincidence detection of Golgi PI4P and the Arf1 GTPase. In addition, PI4P, as substrate for production of phosphatidylinositol 4, 5 phosphate (PIP2), regulates membrane trafficking from the Golgi. We propose that the localization and enzymatic activity of PI4KII? in the Golgi and Golgi-derived organelles are regulated. Recent data from our lab and others show that the cell has at least two populations of PI4KII? that have different intrinsic catalytic activity and differential association with buoyant vs dense membranes fractions. We recently found that PI4KII?'s catalytic activity, its integral membrane association and partitioning into buoyant noncaveolar "rafts" are critically dependent on its palmitoylation. We therefore propose that these functions of PI4KII? are dynamically regulated by reversible palmitoylation of multiple cysteine residues in PI4KII?. We propose four Specific Aims to test this hypothesis. Aim I. Examine the role of PI4KII? in the generation of dynamic Golgi membrane trafficking carriers that contain PI4KII?. We will characterize these carriers and determine if their generation is dependent on the local synthesis of PI4P per se or downstream synthesis of PIP2. Aim II. Examine the role of PI4P in the recruitment of the AP-3 adaptor protein to endomembranes, to evaluate if it, like the related AP-1, binds target membranes through coincidence detection involving PI4P. Aim III. Determine how palmitoylation regulates PI4KII?. The palmitoylacyl transferase that palmitoylates PI4KII? will be identified and the effect of manipulating its expression on PI4KII? behavior will be examined. Aims I-III focus on the export of membranes from Golgi. Aim IV focuses on the recruitment of PI4KII? to the Golgi. Aim IV. Identify PI4KII?'s primary Golgi targeting motif and interactive proteins. We will identify PI4KII? motifs that are necessary and sufficient to direct PI4KII? to the Golgi prior to palmitoylation and will use an integrated proteomics approach to identify PI4KII? Golgi docking proteins and interactive partners. PUBLIC HEALTH RELEVANCE: Membrane phosphoinositides are major regulators of membrane trafficking, and both their synthesis and degradations are required for dynamic membrane movement and vesicle trafficking within the cell. The experiments in this proposal are designed to examine the role of a lipid kinase that makes an essential lipid in the Golgi. Disruption of this kinase or its misregulation will result in trafficking problems that can contribute to the development of multiple metabolic and neurodegenerative diseases due to improper trafficking of essential components within the cell. Problems with this and other related kinases have already been implicated in neurodegenerative diseases, including Alzheimer's disease.

描述（由申请人提供）：我们的总体假设是PI4KII？（磷脂酰肌醇4激酶II？），在高尔基体中产生超过50％的磷脂酰肌醇4磷酸（PI4P），通过其局部生产PI4P来调节高尔基体功能。 PI4P本身通过通过Golgi PI4P和ARF1 GTPase的巧合检测来募集网格蛋白衔接蛋白（例如AP-1）来建立高尔基体的独特细胞器身份。此外，PI4P作为用于产生磷脂酰肌醇4、5磷酸盐（PIP2）的底物，可调节高尔基体膜运输。我们建议PI4KII的定位和酶活性？在高尔基体和高尔基体衍生的细胞器中受到调节。来自我们实验室的最新数据表明，该单元至少有两个PI4KII群体？具有不同的内在催化活性和与浮力与密集膜分数的不同关联。最近，我们发现PI4KII的催化活性，其整体膜关联并分配到浮力非熟练的“筏”中，非常依赖其棕榈酰化。因此，我们建议PI4KII的这些功能？是否通过PI4KII中多个半胱氨酸残基的可逆棕榈酰化动态调节。我们提出了四个特定的目的来检验这一假设。 AIM I.检查PI4KII的作用？在包含pi4kii的动态高尔基膜运输载体的产生中。我们将表征这些载体，并确定它们的产生是否取决于PI4P本身的局部合成或PIP2的下游合成。目标II。检查PI4P在募集AP-3衔接蛋白对子宫内膜的募集中的作用，以评估它是否像相关的AP-1一样，通过涉及PI4P的复合检测来结合靶膜。目标三。确定棕榈酰化如何调节PI4KII？棕榈酰基转移酶是棕榈酰化的PI4KII？会被鉴定，以及操纵其表达对PI4KII的影响吗？行为将被检查。目标I-III专注于高尔基体的膜出口。 AIM IV专注于招募PI4KII？到高尔基。目标IV。识别PI4KII？的主要高尔基体靶向基序和互动蛋白。我们将识别PI4KII？必要且足以指导PI4KII的图案？在棕榈酰化之前到高尔基体，并将使用综合蛋白质组学方法识别PI4KII？高尔基对接蛋白质和互动伙伴。公共卫生相关性：膜磷酸肌醇是膜贩运的主要调节剂，它们的合成和降解都是动态膜运动和细胞内囊泡运输所必需的。该提案中的实验旨在检查脂质激酶在高尔基体中成为必不可少的脂质的作用。这种激酶的破坏或其不调节会导致贩运问题，这可能会导致多种代谢和神经退行性疾病的发展，这是由于细胞内基本成分的不当运输而导致的。这种和其他相关激酶的问题已经与包括阿尔茨海默氏病在内的神经退行性疾病有关。