Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

• 批准号: 8061658
• 负责人: HELEN L YIN
• 金额: $ 40.32万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8061658
• 关键词: 1-Phosphatidylinositol 4-Kinase; Acyltransferase; Adaptor Signaling Protein; Address; Alzheimer' s Disease; Behavior; Binding; Biology; Capsid Proteins; Cells; Clathrin; Clathrin Adaptors; Cysteine; Data; Dependence; Destinations; Detection; Development; Docking; Early Endosome; Endosomes; Epitopes; Generations; Goals; Golgi Apparatus; Golgi Targeting; Grant; Growth Factor; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterodimerization; Hybrids; Immunoprecipitation; Integral Membrane Protein; Isotopes; Life; Lipids; Lysosomes; Medial Golgi; Membrane; Membrane Microdomains; Membrane Protein Traffic; Membrane Proteins; Metabolic Diseases; 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

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50%以上的磷脂酰肌醇4磷酸（PI4P），通过其PI4P的局部产生来调节高尔基体功能。PI4P自身通过高尔基PI4P和Arf1 GTPase的重合检测，募集网格蛋白适配蛋白（如AP-1），从而建立高尔基体独特的细胞器身份。此外，PI4P作为生产磷脂酰肌醇4,5磷酸（PIP2）的底物，调节来自高尔基体的膜运输。我们提出PI4KII？在高尔基和高尔基衍生细胞器中受到调节。我们实验室和其他实验室的最新数据表明，细胞中至少有两个PI4KII？它们具有不同的内在催化活性和与浮力膜和致密膜组分的不同关联。我们最近发现PI4KII？它的催化活性，它的整体膜结合和分配成浮力的非空泡“筏”是严重依赖于它的棕榈酰化。因此，我们提出PI4KII？受PI4KII中多个半胱氨酸残基可逆棕榈酰化的动态调节。我们提出了四个具体目标来检验这一假设。目的一：检验PI4KII的作用？在动态高尔基膜运输载体的产生中含有PI4KII？我们将对这些载体进行表征，并确定它们的产生是否依赖于PI4P本身的局部合成或PIP2的下游合成。目的二世。研究PI4P在AP-3接头蛋白向细胞膜募集中的作用，通过涉及PI4P的一致性检测来评估它是否像相关的AP-1一样结合靶膜。第三目标。确定棕榈酰化如何调节PI4KII？棕榈酰化PI4KII的棕榈酰转移酶？以及调控其表达对PI4KII的影响？行为将受到审查。目的I-III重点关注高尔基体膜的出口。目标IV侧重于PI4KII的招募？到高尔基。目标四：识别PI4KII？主要的高尔基靶基序和相互作用蛋白。我们将确定PI4KII？指导PI4KII的必要和充分的动机？在棕榈酰化之前对高尔基体进行鉴定，并将使用综合蛋白质组学方法鉴定PI4KII？高尔基对接蛋白和相互作用的伙伴。