Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Alan N Hunt,Graeme T Clark,George S Attard,Anthony D Postle

doi:10.1074/jbc.m009878200

Alan N Hunt, Graeme T Clark + Show 2 more

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https://doi.org/10.1074/jbc.m009878200

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Abstract

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase alpha is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

Highlights

A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase ␣ is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ
We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers
Modified Chinese hamster ovary MT 58 cells [13] expressing a temperature-sensitive mutation of CCT␣ are not viable when grown at the restrictive temperature [12], suggesting that endonuclear synthesis of PtdCho is essential for cell survival

Summary

Introduction

The capacity of the nuclear matrix of IMR-32 cells to sustain a compartmentalized synthesis of PtdCho was determined by evaluating the presence, and assaying the activities, of each of the three enzymes of the CDP-choline pathway, namely choline kinase (EC 2.7.1.32), CCT, and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (Table I). The ratio of endogenous to newly synthesized PtdCho molecular species was determined by incubating IMR-32 cells with choline-d9 and extracting the PtdCho from isolated nuclei and from whole cells.

Results

Conclusion