Ultrastructural localization of CTP:phosphoethanolamine cytidylyltransferase in rat liver

CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) (ethanolamine-phosphate cytidylyltransferase, ET) was recently purified to homogeneity from a post-microsomal supernatant of rat liver and subsequently used to raise a polyclonal antibody against the enzyme in rabbits (Vermeulen, 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 immunogold labeling studies on rat liver cryosections. Single-label experiments 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 marginally labeled for ET. Double-label experiments for ET and established markers for either soluble or integral endoplasmic reticulum proteins suggested a bimodal distribution of ET between the RER cisternae and the cytosolic space. Complementary single-label studies for ET and the soluble marker protein showed that the fraction of ET label that was present on RER cisternae was significantly greater than that of the soluble marker, supporting the idea of an uneven distribution. These immunoelectron microscopy studies strongly suggest that the cellular organization of ET differs considerably from that reported recently for the corresponding enzyme in the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase (Wang, Y., Sweiter, T. D., Weinhold, P. A., and Kent, C. (1993) J. Biol. Chem. 268, 5899-5904).