Phosphoinsitide Regulation of the Golgi

描述（由申请人提供）：目前的研究范式表明，在哺乳动物高尔基体中，PI4P主要作为pi4,5p2 （PIP2）的前体，PIP2是一种重要的质膜调节剂。我们发现PI4P在哺乳动物高尔基体中富集，并利用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。pi4kii α RNAi降低高尔基PI4P，阻断网格蛋白接头AP-1复合物向TGN的募集，也阻断AP-1从TGN独立输出组成性分泌蛋白。通过添加PI4P而不是PIP2来修复AP-1的募集缺陷。此外，纯化的AP-1与PI4P结合，抗PI4P抑制胞质AP-1向正常细胞膜的体外募集。我们提出(i) pi4kii α通过产生pi4p -富结构域（指定AP-1外壳机制的Arf招募）建立高尔基体独特的脂质定义细胞器身份；（ii） pi4kii α通过支持PIP2合成来调节组成型分泌，因为pi4kii α RNAi细胞的VSVG输出缺陷可以通过添加回PI4P或PIP2来修复。目的一：确定AP-1是否通过与PI4P结合介导向高尔基体募集。AP-1 I μ亚基中潜在的PI4P结合残基将发生突变，这些突变的功能后果将在体内和体外进行研究。体内试验包括确定突变亚基是否降低了挽救mu 1 -/-成纤维细胞高尔基表型的能力。目的二世。研究PI4P在调节其他外壳蛋白的高尔基募集中的作用，并使用延时荧光成像来表征PI4P水平变化对tgn衍生转运载体动态行为的影响。第三目标。确定在AP-1募集和VSVG输出中，arf募集的高尔基PI4K是否与pi4kii α功能重叠。pi4kiil β将被过表达以确定它是否拯救pi4kii α RNAi高尔基表型，pi4kiil β将被RNAi敲低以确定它是否与pi4kii α RNAi在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 (pi5pkβ)是否调节这些高尔基PI4Ks下游的VSVG输出。

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.