Helms J B, de Vries K J, Wirtz K W
Center for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands.
J Biol Chem. 1991 Nov 15;266(32):21368-74.
We have investigated the intracellular localization and synthesis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in Chinese hamster ovary (CHO) cells by analyzing membrane fractions that were obtained by sucrose density gradient centrifugation. After labeling the cells for 24 h with [3H]inositol, the bulk of [3H] PtdInsP2 was found in the plasma membrane fraction, yet this lipid was also distinctly present in the microsomal fraction (20% of total cellular [3H]PtdInsP2). To determine the origin of this microsomal PtdInsP2, gradient fractions from unlabeled CHO cells were incubated with [3H]inositol in the presence of an ATP-generating system. Under these conditions of labeling, [3H]PtdIns was exclusively present in the microsomal fractions and found to be partially converted to [3H] phosphatidylinositol 4-phosphate ([3H]PtdInsP) and phosphatidylinositol 4-phosphate ([3H]PtdInsP) and [3H]PtdInsP2. The ability of microsomes to synthesize PtdInsP and PtdInsP2 was confirmed by assaying the gradient fractions for PtdIns and PtdInsP kinase activity using endogenous substrate and [gamma-32P]ATP. In the presence of exogenous substrate and Triton X-100, PtdInsP kinase activity was particularly high in the plasma membrane fractions. When phosphoinositide synthesis was studied in permeabilized CHO cells under conditions of sustained membrane vesicle flow (Helms, J. B., Karrenbauer, A., Wirtz, K. W. A., Rothman, J. E., and Wieland, F. T. (1990) J. Biol. Chem. 265, 20027-20032), no lag-time could be detected between the synthesis of [3H]PtdIns and the formation of [3H]PtdInsP2. Moreover, when lipid transport pathways were blocked in these permeabilized cells either by omission of membrane-free cytosol, addition of GTP gamma S and brefeldin A, or temperature block, PtdInsP2 formation still occurred at normal levels. These results strongly suggest that PtdInsP2 can be formed at the site of PtdIns synthesis, i.e. the endoplasmic reticulum (ER). The relationship between PtdInsP2, generated in the ER, and PtdInsP2 present in the plasma membrane, remains to be established.
我们通过分析蔗糖密度梯度离心获得的膜组分，研究了中国仓鼠卵巢（CHO）细胞中磷脂酰肌醇4,5 - 二磷酸（PtdInsP2）的细胞内定位和合成。用[3H]肌醇标记细胞24小时后，大部分[3H]PtdInsP2存在于质膜组分中，但这种脂质也明显存在于微粒体组分中（占细胞总[3H]PtdInsP2的20%）。为了确定这种微粒体PtdInsP2的来源，将未标记的CHO细胞的梯度组分在有ATP生成系统存在的情况下与[3H]肌醇一起孵育。在这些标记条件下，[3H]PtdIns仅存在于微粒体组分中，并发现其部分转化为[3H]磷脂酰肌醇4 - 磷酸（[3H]PtdInsP）和[3H]磷脂酰肌醇4,5 - 二磷酸（[3H]PtdInsP2）。通过使用内源性底物和[γ-32P]ATP测定梯度组分中的PtdIns和PtdInsP激酶活性，证实了微粒体合成PtdInsP和PtdInsP2的能力。在存在外源性底物和Triton X - 100的情况下，质膜组分中的PtdInsP激酶活性特别高。当在持续膜泡流动的条件下（Helms, J. B., Karrenbauer, A., Wirtz, K. W. A., Rothman, J. E., and Wieland, F. T. (1990) J. Biol. Chem. 265, 20027 - 20032）研究通透化CHO细胞中的磷酸肌醇合成时，在[3H]PtdIns的合成与[3H]PtdInsP2的形成之间未检测到延迟时间。此外，当通过省略无膜胞质溶胶、添加GTPγS和布雷菲德菌素A或温度阻断来阻断这些通透化细胞中的脂质转运途径时，PtdInsP2的形成仍以正常水平发生。这些结果强烈表明PtdInsP2可以在PtdIns合成的部位即内质网（ER）形成。在内质网中生成的PtdInsP2与质膜中存在的PtdInsP2之间的关系仍有待确定。
