Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

• 批准号: 7215567
• 负责人: HELEN L YIN
• 金额: $ 28.47万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215567
• 关键词: 1-Phosphatidylinositol 4-Kinase; Adaptor Protein Complex 1; Back; Behavior; Binding; Biogenesis; Biological Assay; Capsid Proteins; Cell membrane; Cells; Cellular Membrane; Clathrin Adaptors; Complex; Defect; Enzymes; Exocytosis; Fibroblasts; Golgi Apparatus; Grant; In Vitro; Life; Lipids; Mediating; Membrane; Membrane Protein Traffic; Metabolism; Mutate; Mutation; Organelles; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Protein Overexpression; Proteins; RNA Interference; Recruitment Activity; Regulation; Role; Sorting - Cell Movement; Specific qualifier value; Stomatitis; Testing; Viral Proteins; Yeasts; cofactor; fluorescence imaging; in vivo; insight; knock-down; mutant; phosphatidylinositol 4-phosphate; prevent; time use

DESCRIPTION (provided by applicant): Current paradigms propose that in the mammalian Golgi, PI4P acts primarily as a precursor to PI4,5P2 (PIP2), which is an important plasma membrane regulator. We found that PI4P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase (PI4K), to determine if PI4P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI4P, blocks the recruitment of clathrin adaptor AP-1 complexes to the TGN and also blocks AP-1 independent export of constitutively secreted proteins from the TGN. The AP-1 recruitment defect is rescued by adding back PI4P but not PIP2. In addition, purified AP-1 binds PI4P, and anti-PI4P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that (i) PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI4P-rich domains that specify Arf recruitment of the AP-1 coat machinery; (ii) PI4KIIalpha regulates constitutive secretion by supporting PIP2 synthesis, because the VSVG export defect in PI4KIIalpha RNAi cells can be rescued by adding back either PI4P or PIP2. Aim I. Determine if AP-1 recruitment to the Golgi is mediated through AP-1 binding to PI4P. Potential PI4P binding residues in the AP-1 I mu subunit will be mutated, and the functional consequences of these mutations will be examined in vivo and in vitro. In vivo assays include determining if the mutant subunit has diminished ability to rescue the Golgi phenotype of mu 1 -/- fibroblasts. Aim II. Examine the role of PI4P in regulating the Golgi recruitment of other coat proteins and use time-lapse fluorescence imaging to characterize the effects of changing PI4P levels on the dynamic behavior of TGN-derived transport carriers. Aim III. Determine if PI4KIIlbeta, an Arfrecruited Golgi PI4K, overlaps functionally with PI4KIIalpha in AP-1 recruitment and VSVG export. PI4KIIlbeta will be overexpressed to determine if it rescues the PI4KIIalpha RNAi Golgi phenotypes, and PI4KIIlbeta will be knocked down by RNAi to determine if it inhibits secretion at the same or a different step as PI4KIIalpha RNAi. We will determine if phosphatidylinositol 4 phosphate 5 kinase beta (PI5PKbeta) regulates VSVG export downstream of these Golgi PI4Ks.

描述（由申请人提供）：当前范例提出，在哺乳动物高尔基体中，PI4P 主要充当 PI4,5P2 (PIP2) 的前体，PI4,5P2 (PIP2) 是重要的质膜调节剂。我们发现 PI4P 在哺乳动物高尔基体中富集，并使用 PI4KIIalpha（高尔基体驻留磷脂酰肌醇 4 激酶 (PI4K)）的 RNA 干扰 (RNAi) 来确定 PI4P 是否直接调节高尔基体。 PI4KIIalpha RNAi 降低高尔基体 PI4P，阻止网格蛋白接头 AP-1 复合物向 TGN 的募集，还阻止 AP-1 独立地从 TGN 输出组成型分泌蛋白。 AP-1 募集缺陷可以通过添加回 PI4P 而不是 PIP2 来弥补。此外，纯化的 AP-1 可以结合 PI4P，而抗 PI4P 可以在体外抑制胞质 AP-1 向正常细胞膜的募集。我们建议 (i) PI4KIIalpha 通过生成富含 PI4P 的结构域来建立高尔基体独特的脂质定义的细胞器身份，这些结构域指定 AP-1 外壳机制的 Arf 募集； (ii) PI4KIIalpha 通过支持 PIP2 合成来调节组成型分泌，因为 PI4KIIalpha RNAi 细胞中的 VSVG 输出缺陷可以通过添加回 PI4P 或 PIP2 来挽救。目标 I. 确定 AP-1 招募到高尔基体是否是通过 AP-1 与 PI4P 结合介导的。 AP-1 I mu 亚基中潜在的 PI4P 结合残基将发生突变，并且将在体内和体外检查这些突变的功能后果。体内测定包括确定突变亚基是否降低了拯救 mu 1 -/- 成纤维细胞高尔基体表型的能力。目标二。检查 PI4P 在调节高尔基体招募其他外壳蛋白中的作用，并使用延时荧光成像来表征改变 PI4P 水平对 TGN 衍生转运载体动态行为的影响。目标三。确定 PI4KIIlbeta（一种 Arfrecruited 高尔基体 PI4K）在 AP-1 募集和 VSVG 输出中是否与 PI4KIIalpha 功能重叠。 PI4KII1β将被过表达以确定其是否拯救PI4KIIα RNAi高尔基体表型，并且PI4KII1β将被RNAi敲低以确定其是否在与PI4KIIα RNAi相同或不同的步骤抑制分泌。我们将确定磷脂酰肌醇 4 磷酸 5 激酶 β (PI5PKbeta) 是否调节这些高尔基体 PI4K 下游的 VSVG 输出。