Phosphoinsitide Regulation of the Golgi

描述(申请人提供)：我们的总体假设是PI4KII？在高尔基体中产生50%以上的磷脂酰肌醇4磷酸(PI4P)的磷脂酰肌醇4激酶II？，通过其局部产生PI4P来调节高尔基体的功能。PI4P本身通过对高尔基体PI4P和Arf1 GTP酶的重合检测招募笼状蛋白适配器蛋白(如AP-1)来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。我们认为PI4KII？在高尔基体和高尔基体衍生的细胞器中是受调控的。来自我们实验室和其他实验室的最新数据显示，该细胞至少有两个PI4KII？具有不同的固有催化活性和与浮膜组分和致密膜组分的不同缔合。我们最近发现，PI4KII？的S催化活性、其整膜结合和分配成浮力的非空泡“筏”严重依赖于它的棕榈酰化作用。因此，我们提出PI4KII的这些功能？受PI4KII中多个半胱氨酸残基的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。目标1.研究PI4KII的作用？在含有PI4KII？的动态高尔基体膜运输载体的产生中。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II.检测PI4P在AP-3接头蛋白募集到内膜中的作用，评价其是否与相关的AP-1一样，通过与PI4P的符合检测而与靶膜结合。目的III.确定棕榈酰化如何调节PI4KII？棕榈酰化PI4KII的棕榈酰转移酶？是否会被鉴定，以及操纵其表达对PI4KII的影响？我们将检查他们的行为。AIMS I-III的重点是高尔基地区的膜出口。目标四侧重于招募PI4KII？去高尔基山脉。目的IV.鉴定PI4KII？S的主要高尔基体靶向基序和相互作用蛋白。我们会确认PI4KII的身份吗？对于导演PI4KII来说，哪些主题是必要的和充分的？并将使用整合的蛋白质组学方法来鉴定PI4KII？高尔基体对接蛋白质和互动伙伴。 与公共健康相关：膜磷脂是膜运输的主要调节者，它们的合成和降解都是细胞内动态膜运动和囊泡运输所必需的。这项建议中的实验旨在研究一种脂激酶的作用，它是高尔基体中一种必不可少的脂类。该激酶的破坏或其调控不当将导致运输问题，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造成的。这种和其他相关的蛋白激酶的问题已经被认为与神经退行性疾病有关，包括阿尔茨海默病。

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.