Document Detail


Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types.
MedLine Citation:
PMID:  10918057     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
C J DeLong; L Qin; Z Cui
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  275     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2000 Oct 
Date Detail:
Created Date:  2000-11-13     Completed Date:  2000-11-13     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  32325-30     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, Wake Forest University, School of Medicine, Winston-Salem, North Carolina 27157, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Catalysis
Cell Cycle*
Cell Line
Cell Nucleus / drug effects,  enzymology*,  metabolism
Choline-Phosphate Cytidylyltransferase / chemistry,  genetics,  metabolism*
Culture Media, Serum-Free
Cytoplasm / drug effects,  metabolism
Flow Cytometry
Green Fluorescent Proteins
Humans
Luminescent Proteins
Microscopy, Fluorescence
Mutation / genetics
Nuclear Localization Signals / genetics,  physiology
Oleic Acid / pharmacology
Organ Specificity
Phosphatidylcholines / biosynthesis,  metabolism
Protein Transport / drug effects
Recombinant Fusion Proteins / chemistry,  genetics,  metabolism
Temperature
Transfection
Grant Support
ID/Acronym/Agency:
CA-09422/CA/NCI NIH HHS; R01-CA79670/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Culture Media, Serum-Free; 0/Luminescent Proteins; 0/Nuclear Localization Signals; 0/Phosphatidylcholines; 0/Recombinant Fusion Proteins; 112-80-1/Oleic Acid; 147336-22-9/Green Fluorescent Proteins; EC 2.7.7.15/Choline-Phosphate Cytidylyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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