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

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CT alpha), this study was designed to visualize green fluorescent protein (GFP).CT alpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP.CT alpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP-CT alpha 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 CT alpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CT alpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CT alpha 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 CT alpha. The GFP.CT alpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CT alpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CT alpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.