Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

• 批准号: 7048650
• 负责人: HELEN L YIN
• 金额: $ 29.32万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048650
• 关键词: Golgi apparatus; RNA interference; binding sites; clathrin; confocal scanning microscopy; conformation; laboratory rabbit; liposomes; phenotype; phosphatidylinositols; phosphorylation; protein binding; protein isoforms; protein protein interaction; protein structure function; time resolved data; transcription factor

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）：目前的范例提出，在哺乳动物高尔基体中，PI 4P主要作为PI 4，5 P2（PIP 2）的前体，PI 4，5 P2是一种重要的质膜调节剂。我们发现PI 4P在哺乳动物高尔基体中富集，并使用RNA干扰（RNAi）的PI 4KII α，高尔基体居民磷脂酰肌醇4激酶（PI 4K），以确定是否PI 4P直接调节高尔基体。PI 4KII α RNAi降低高尔基体PI 4P，阻断网格蛋白衔接子AP-1复合物向TGN的募集，并且还阻断组成型分泌的蛋白质从TGN的AP-1非依赖性输出。通过添加回PI 4P而不是PIP 2来挽救AP-1募集缺陷。此外，纯化的AP-1结合PI 4P，并且抗PI 4P抑制胞质AP-1向正常细胞膜的体外募集。我们提出：（i）PI 4KII alpha通过产生富含PI 4P的结构域来确定高尔基体独特的脂质定义的细胞器身份，该结构域指定AP-1外壳机器的Arf募集;（ii）PI 4KII alpha通过支持PIP 2合成来调节组成性分泌，因为PI 4KII alpha RNAi细胞中的VSVG输出缺陷可以通过添加回PI 4P或PIP 2来挽救。艾姆岛确定AP-1向高尔基体的募集是否通过AP-1与PI 4P结合介导。AP-1 I mu亚基中潜在的PI 4P结合残基将被突变，这些突变的功能后果将在体内和体外进行检查。体内测定包括确定突变亚基是否具有降低的拯救mu 1 -/-成纤维细胞的高尔基体表型的能力。Aim II.检查PI 4P在调节其他外壳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改变PI 4P水平对TGN衍生的运输载体的动态行为的影响。Aim III.确定PI 4KII 1 β（一种Arf募集的高尔基体PI 4K）在AP-1募集和VSVG导出中是否与PI 4KII 1 α功能重叠。PI 4KIII β将被过表达以确定其是否挽救PI 4KII α RNAi高尔基体表型，并且PI 4KIII β将被RNAi敲低以确定其是否在与PI 4KII α 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（PI 5 PK β）是否调节这些高尔基体PI 4Ks下游的VSVG输出。