Synthesis of phosphatidylinositol 4,5-bisphosphate in the endoplasmic reticulum of Chinese hamster ovary cells.

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.