ULTRASTRUCTURAL-LOCALIZATION OF CTP-PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE IN RAT-LIVER

CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) (ethanolami ne-phosphate cytidylyltransferase, ET) was recently purified to homoge neity from a post-microsomal supernatant of rat liver and subsequently used to raise a polyclonal antibody against the enzyme in rabbits (Ve rmeulen, P. S., Tijburg, L. B. M., Geelen, M. J. H., and van Golde, L. M. G. (1993) J. Biol. Chem 268, 7458-7464). In the present study, we used the affinity-purified antibody against ET for ultrastructural imm unogold labeling studies on rat liver cryosections. Single-label exper iments clearly demonstrated that ET label was not randomly distributed in hepatocytes. The ET label was concentrated in areas that contained cisternae of the rough endoplasmic reticulum, whereas other cellular organelles (nuclei, mitochondria, and peroxisomes) were only marginall y labeled for ET. Double-label experiments for ET and established mark ers for either soluble or integral endoplasmic reticulum proteins sugg ested a bimodal distribution of ET between the RER cisternae and the c ytosolic space. Complementary single-label studies for ET and the solu ble marker protein showed that the fraction of ET label that was prese nt on RER cisternae was significantly greater than that of the soluble marker, supporting the idea of an uneven distribution. These immunoel ectron microscopy studies strongly suggest that the cellular organizat ion of ET differs considerably from that reported recently for the cor responding enzyme in the CDP-choline pathway, CTP:phosphocholine cytid ylyltransferase (Wang, Y., Sweiter T. D., Weinhold, P. A., and Kent, C . (1993) J. Biol. Chem. 268, 5899-5904).