Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells.
The location of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary (CHO) cells made deficient in phosphatidylcholine was determined by immunofluorescence techniques. A rabbit polyclonal antibody was raised against a synthetic peptide corresponding to the amino-terminal 17 amino acid residues of rat liver cytidylyltransferase. The antibody recognized both native and denatured cytidylyltransferase from both rat liver and CHO cells. CHO cells were treated with phospholipase C to alter the lipid composition of the plasma membrane and to elicit translocation of cytidylyltransferase from the less active soluble pool to an activated membrane fraction. Visualization of cytidylyltransferase by indirect immunofluorescence revealed staining of the nuclear envelope in phospholipase C-treated cells but not in untreated cells. CHO cells were also starved for choline and supplemented with a choline analogue to provide an alternative technique of rendering the cells phosphatidylcholine-deficient. Although this treatment should affect different cellular membranes than those affected by phospholipase C treatment, cytidylyltransferase still translocated to the nuclear envelope, as shown by indirect immunofluorescence. These results indicate that activated, membrane-bound cytidylyltransferase is associated with the nuclear membrane and suggest that the nuclear membrane may be a site of de novo phosphatidylcholine synthesis.
Watkins
J D
JD
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Kent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology*; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D014644:Genetic Variation; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010713:Phosphatidylcholines / Q000378:metabolism*; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism
D000906:Antibodies; D010455:Peptides; D010713:Phosphatidylcholines; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D010738:Type C Phospholipases