Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

• 批准号: 7465179
• 负责人: HELEN L YIN
• 金额: $ 31.81万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7465179
• 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; Disruption; 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; Localized; 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; Protein Overexpression; Proteins; Proteomics; Public Health; RID; 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; palmitoylation; phosphatidylinositol 4-phosphate; protein protein interaction; 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？）在高尔基体中产生超过 50% 的磷脂酰肌醇 4 磷酸 (PI4P)，通过其局部产生 PI4P 来调节高尔基体功能。 PI4P 本身通过高尔基体 PI4P 和 Arf1 GTPase 的一致性检测招募网格蛋白接头蛋白（例如 AP-1）来建立高尔基体独特的细胞器身份。此外，PI4P 作为产生磷脂酰肌醇 4, 5 磷酸 (PIP2) 的底物，调节高尔基体的膜运输。我们提出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。我们实验室和其他实验室的最新数据表明，该细胞至少有两个 PI4KII 群体？具有不同的内在催化活性以及与浮力膜和致密膜部分的不同关联。我们最近发现 PI4KII 的催化活性、其完整的膜缔合和划分成浮力非空穴“筏”主要取决于其棕榈酰化。因此我们提出PI4KII的这些功能？通过 PI4KII? 中多个半胱氨酸残基的可逆棕榈酰化来动态调节。我们提出四个具体目标来检验这一假设。目标 I. 检查 PI4KII 的作用？包含 PI4KII 的动态高尔基体膜运输载体的生成？我们将表征这些载体并确定它们的生成是否依赖于 PI4P 本身的局部合成或 PIP2 的下游合成。目标二。检查 PI4P 在 AP-3 接头蛋白募集到内膜中的作用，以评估它是否像相关的 AP-1 一样，通过涉及 PI4P 的符合检测结合靶膜。目标三。确定棕榈酰化如何调节 PI4KII？棕榈酰基转移酶可棕榈酰化 PI4KII？将被识别并操纵其表达对 PI4KII 的影响？行为将受到检查。目标 I-III 重点关注高尔基体膜的输出。 Aim IV 重点招募 PI4KII？到高尔基体。目标四。识别 PI4KII？的主要高尔基体靶向基序和相互作用蛋白。我们会识别PI4KII吗？指导 PI4KII 所必需且充分的基序？在棕榈酰化之前先检测高尔基体并使用综合蛋白质组学方法来识别 PI4KII？高尔基对接蛋白和互动伙伴。 公共卫生相关性：膜磷酸肌醇是膜运输的主要调节剂，它们的合成和降解是细胞内动态膜运动和囊泡运输所必需的。该提案中的实验旨在检查脂质激酶的作用，该脂质激酶在高尔基体中形成必需脂质。这种激酶的破坏或其失调将导致运输问题，由于细胞内重要成分的运输不当，可能导致多种代谢和神经退行性疾病的发展。这种激酶和其他相关激酶的问题已经与神经退行性疾病有关，包括阿尔茨海默病。