title: CTP:phosphocholine cytidylyltransferase α (CCTα) and lamins alter nuclear membrane structure without affecting phosphatidylcholine synthesis.
abstract: CTP:phosphocholine cytidylyltransferase α (CCTα) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway for phosphatidylcholine (PC) synthesis. Lipid activation of CCTα 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 CCTα 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, CCTα was nucleoplasmic and co-localized with GFP-progerin in nuclear folds and invaginations; however, HGPS fibroblasts displayed an abnormal distribution of CCTα 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 CCTα 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.
authors.affiliation: Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Karsten
authors.initials: K
authors.lastname: Gehrig
authors.forename: Neale D
authors.initials: ND
authors.lastname: Ridgway
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism; D034921:Lamin Type B / Q000235:genetics / Q000378:metabolism; D034882:Lamins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D034881:Nuclear Lamina / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D011371:Progeria / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism
chemical_list: D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D010713:Phosphatidylcholines; D049452:Green Fluorescent Proteins; D019301:Oleic Acid; D019916:Choline-Phosphate Cytidylyltransferase; C517213:PCYT1A protein, human; D017855:Transferases (Other Substituted Phosphate Groups); C119514:choline-ethanolaminephosphotransferase; D002794:Choline