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Journal of Lipid Research, Vol. 50, 2182-2192, November 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex

Nicole Testerink, Michiel H. M. van der Sanden, Martin Houweling, J. Bernd Helms and Arie B. Vaandrager¹

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, and Institute of Biomembranes, University of Utrecht, 3584 CM, The Netherlands

¹ To whom correspondence should be addressed. e-mail: a.b.vaandrager{at}uu.nl

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40°C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [³H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.

Supplementary key words CTP:phosphocholine cytidylyltransferase • lipid droplets • protein transport • diacylglycerol

Abbreviations: CHO, Chinese hamster ovary; CT, CTP:phosphocholine cytidylyltransferase; DAG, diacylglycerol; EM, electron microscopy; ER, endoplasmic reticulum; FRAP, fluorescence recovery after photobleaching; LD, lipid droplet; lysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; PKD, protein kinase D; pVSVG-GFP, plasmid containing the coding sequence for vesicular stomatitis virus glycoprotein fused to green fluorescent protein; RLU, relative light units; TAG, triacylglycerol; TGN, trans-Golgi network; YFP, yellow fluorescent protein

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