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）：当前范式提出，在哺乳动物的高尔基体中，PI4P主要充当PI4,5P2（PIP2）的前体，这是重要的质膜调节剂。我们发现PI4P富含哺乳动物的高尔基体，并使用Golgi常驻磷脂酰肌醇4激酶（PI4K）PI4Kiialpha的RNA干扰（RNAi）来确定PI4P是否直接调节了高尔基体。 pi4kiialpha rNAi降低了高尔基体PI4P，阻止了网格蛋白适配器AP-1复合物的募集到TGN，并阻止了从TGN中独立于AP-1独立出口的组成型蛋白质。 AP-1招聘缺陷是通过添加PI4P而非PIP2来挽救的。此外，纯化的AP-1结合PI4P，抗PI4P抑制胞质AP-1的体外募集到正常的细胞膜。我们建议（i）pi4kiialpha通过生成指定ARF募集AP-1涂层机械的富含PI4P的域来建立Golgi独特的脂质定义的细胞器身份； （ii）PI4Kiialpha通过支持PIP2合成来调节本构分泌，因为可以通过添加PI4P或PIP2来挽救PI4Kiialpha RNAi细胞中的VSVG输出缺陷。 AIM I.确定AP-1募集到高尔基体是否是通过与PI4P结合的AP-1介导的。 AP-1 I MU亚基中的潜在PI4P结合残基将被突变，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-/ - 成纤维细胞的高尔基表型的能力。目标II。检查PI4P在调节其他外套蛋白的高尔基体募集中的作用，并使用延时荧光成像来表征PI4P水平改变PI4P水平对TGN衍生传输载体动态行为的影响。目标三。确定Pi4Kiilbeta是一种arfrecrucrue的高尔基pi4k，在AP-1募集中与PI4Kiialpha重叠，并在AP-1募集中重叠。 PI4Kiilbeta将过表达，以确定它是否挽救了Pi4kiialpha RNAi Golgi表型，并且RNAi将击倒Pi4kiilbeta，以确定它是否抑制分泌在同一或不同的步骤中，或者是不同的步骤，或者是不同的步骤。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PI4K的下游VSVG导出。