Intracellular localization of phospholipase D1 in mammalian cells
Intracellular localization of phospholipase D1 in mammalian cells
复制标题
DOI：
10.1091/mbc.12.4.943
复制
发表时间：
2001-04-01
影响因子：
3.3
通讯作者：
Shields, D
中科院分区：
文献类型：
--
作者：
Freyberg, Z;Sweeney, D;Shields, D
磷脂酶D（PLD）水解磷脂酰胆碱以产生磷脂酸。在哺乳动物细胞中，这种反应与外被体向高尔基体膜的募集和新生分泌囊泡从高尔基体网络中的释放有关。这些观察结果表明，PLD是与高尔基复合体，然而，迄今为止，由于其丰度低，PLD的细胞内定位的特点是只能间接通过嵌合蛋白的过表达。我们已经使用了高灵敏度的抗体PLD1连同免疫荧光和免疫金电子显微镜以及细胞分级，以确定内源性PLD1在几种细胞类型的细胞内定位。尽管PLD 1具有弥散染色模式，但其在高尔基体中显著富集，并且也存在于细胞核中。在高尔基体的碎片与诺考达唑治疗，PLD 1密切相关的膜片段，而PA合成抑制后，PLD 1从膜上解离。血凝素标记形式的PLD 1的过表达导致内源性酶从其核周定位到大的囊泡结构的位移。令人惊讶的是，当高尔基体崩溃，响应布雷菲德菌素A，核定位的PLD 1显着增强。我们的数据表明，PLD 1的细胞内定位与从高尔基体运输囊泡的作用一致，并表明它也在细胞核中发挥作用。
Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.