Phosphoinsitide Regulation of the Golgi

描述(申请人提供)：目前的范例表明，在哺乳动物高尔基体中，PI4P主要作为PI4，5P2(PIP2)的前体，PI4，5P2是一种重要的质膜调节因子。我们发现PI4P在哺乳动物高尔基体中含量丰富，并利用高尔基体驻留的磷脂酰肌醇4激酶(PI4K)PI4KIIpha的RNA干扰(RNAi)来确定PI4P是否直接调节高尔基体。PI4KIIpha RNAi可降低高尔基体PI4P，阻断网状蛋白适配器AP-1复合体向TGN的募集，并阻止AP-1独立地将构成分泌的蛋白质从TGN输出。AP-1招募缺陷通过重新添加PI4P而不是PIP2来修复。此外，纯化的AP-1与PI4P结合，而抗PI4P可抑制胞浆AP-1在体外对正常细胞膜的募集。我们认为(I)PI4KIIpha通过产生PI4P丰富的结构域，指定AP-1涂层机制的Arf招募，建立了高尔基体独特的脂质定义细胞器的身份；(Ii)PI4KIIpha通过支持PIP2的合成来调节结构性分泌，因为PI4KIIpha RNAi细胞中的VSVG输出缺陷可以通过重新添加PI4P或PIP2来挽救。目的I.确定AP-1是否通过AP-1与PI4P结合而向高尔基体募集。AP-1 Iu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检测。体内试验包括确定突变的亚基是否降低了挽救mu1-/-成纤维细胞高尔基表型的能力。目的II.研究PI4P在调节其他外壳蛋白高尔基体募集中的作用，并利用时间推移荧光成像来表征PI4P水平的变化对TGN来源的运输载体动态行为的影响。目的III.确定在AP-1招募和VSVG输出中，Arf招募的高尔基PI4K是否与PI4KIIpha在功能上重叠。PI4KIIlbeta将被过度表达，以确定它是否拯救PI4KIIpha RNAi高尔基表型，PI4KIIlbeta将被RNAi击倒，以确定它是否在与PI4KIIpha RNAi相同或不同的步骤中抑制分泌。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。

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.