Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells.
Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.7.15), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.
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INSERM Unité 326, Hôpital Purpan, Toulouse, France.
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D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D003994:Bucladesine / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002794:Choline / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase; D003600:Cytosol / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D051379:Mice; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019319:Okadaic Acid; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000096:biosynthesis; D010743:Phospholipids / Q000378:metabolism
D004988:Ethers, Cyclic; D009829:Oleic Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; D019319:Okadaic Acid; D019301:Oleic Acid; D003994:Bucladesine; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D002794:Choline