Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha 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 (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha 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 CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha 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 CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.
Department of Biochemistry, Wake Forest University, School of Medicine, Winston-Salem, North Carolina 27157, USA.
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D000818:Animals; D002384:Catalysis; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology; D019301:Oleic Acid / Q000494:pharmacology; D009928:Organ Specificity; D010713:Phosphatidylcholines / Q000096:biosynthesis / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D016895:Culture Media, Serum-Free; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019301:Oleic Acid; D019916:Choline-Phosphate Cytidylyltransferase