Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase α fusion protein is independent of cell cycle conditions and cell types

Abstract

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase α (CTα), this study was designed to visualize green fluorescent protein (GFP)·CTα fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP·CTα fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP·CTα fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTα between the nucleus and cytoplasm. Moreover, the subcellular localization of CTα was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTα between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTα. The GFP·CTα lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP·CTα fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTα is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.