CTP:phosphocholine cytidylyltransferase α (CCTα) and lamins alter nuclear membrane structure without affecting phosphatidylcholine synthesis

CTP:phosphocholine cytidylyltransferase alpha (CCT-alpha) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP choline pathway for phosphatidylcholine (PC) synthesis. Lipid activation of CCT alpha results in its translocation to the nuclear envelope and expansion of an intranuclear membrane network termed the nucleoplasmic reticulum (NR) by a mechanism involving membrane deformation. Nuclear lamins are also required for stability and proliferation of the NR, but whether this unique structure, or the nuclear lamina in general, is required for PC synthesis is not known. To examine this relationship, the nuclear lamina was depleted by RNAi or disrupted by expression of the Hutchinson Gilford progeria syndrome (HGPS) mutant lamin A (progerin), and the effect on CCT alpha and choline metabolism was analyzed. siRNA-mediated silencing of lamin A/C or lamin B1 in CHO cells to diminish the NR had no effect on PC synthesis, while double knockdown non-specifically inhibited the pathway. Confirming this minor role in PC synthesis, only 10% of transiently overexpressed choline/ethanolamine phosphotransferase was detected in the NR. In CHO cells, CCT alpha was nucleoplasmic and co-localized with GFP-progerin in nuclear folds and invaginations; however, HGPS fibroblasts displayed an abnormal distribution of CCT alpha in the cytoplasm and nuclear envelope that was accompanied by a 2-fold reduction in PC synthesis. In spite of its altered localization, choline-labeling experiments showed that CCT activity was unaffected, and inhibition of PC synthesis was traced to reduced activity of a hemicholinium-sensitive choline transporter. We conclude that CCT alpha and lamins specifically cooperate to form the NR, but the overall structure of the nuclear envelope has a minimal impact on CCT activity and PC synthesis. (C) 2011 Elsevier B.V. All rights reserved.