Localization of CTP: Phosphocholine cytidylyltransferase in Chinese hamster ovary cells

Janice Nan Morand, Purdue University

Abstract

In Chinese hamster ovary (CHO) cells the rate of phosphatidylcholine biosynthesis is modulated by changes in the amount of membrane-associated CTP: phosphocholine cytidylyltransferase. To begin to understand the mechanism of this process, the subcellular localization of the membrane-associated cytidylyltransferase was determined. A method was developed for the rapid, analytical fractionation of entire CHO cell homogenates on Percoll density gradients. A key feature of this procedure is the addition to the homogenate of either micrococcal nuclease or DNase I in excess of cellular actin. In CHO cells with an altered membrane composition, 80% of the cytidylyltransferase is associated with the membrane. Homogenates from these cells were fractionated on Percoll density gradients and marker enzyme activities were used to determine the location of the cellular membrane fractions. The peak of cytidylyltransferase activity occurred in the gradient at a density intermediate to the peak of the endoplasmic reticulum and plasma membrane markers. The profile of the cytidylyltransferase activity most closely resembled that of the Golgi membrane marker; however, sucrose gradient centrifugation separated the Golgi from the cytidylyltransferase activity. Differential centrifugation suggested a nuclear membrane association of the enzyme. Cytidylyltransferase was associated with a membrane fraction that sedimented when subjected to low speed centrifugation (65 $\times$ g, 5 min). From Percoll gradient fractions, nuclear membranes were identified by microscopy and these membranes migrated with the cytidylyltransferase activity. The data are consistent with a localization of cytidylyltransferase in the nuclear membrane. Antibodies to purified cytidylyltransferase were prepared in chickens. The antibodies reacted with cytidylyltransferase on Western blots and precipitated activity from rat liver and CHO cell cytosol. Immunolocalization of the enzyme in intact cells was attempted. A CHO cell mutant defective in endosome acidification was used to determine if organelle acidification plays a role in the response of cytidylyltransferase to phosphatidylcholine hydrolysis. The cells were impaired in phospholipase C-stimulated phosphatidylcholine synthesis; however, activation and translocation of cytidylyltransferase was unchanged.

Degree

Ph.D.

Advisors

Kent, Purdue University.

Subject Area

Biochemistry

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