Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase.
CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.
Wang
Y
Y
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor 48109-0606.
MacDonald
J I
JI
Kent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002467:Cell Nucleus / Q000201:enzymology*; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D017931:DNA Primers; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D051381:Rats; D014162:Transfection
D017931:DNA Primers; D021382:Protein Sorting Signals; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase