The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions Nicolás O. Favale, María C. Fernández-Tome, Lucila G. Pescio, Norma B. Sterin-Speziale ⁎ Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, IQUIFIB-CONICET, Ciudad Autónoma de Buenos Aires (C1113AAD), Argentina abstract article info Article history: Received 30 November 2009 Received in revised form 2 July 2010 Accepted 12 July 2010 Available online 17 July 2010 Keywords: Nuclear organization CTP:Phosphocholine Cytidylyltransferase Speckle Hypertonic stress Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes. It is predominantly synthesised in mammalian cells and tissue by the CDP-choline pathway [1]. There are three steps in the CDP-choline pathway: choline is first phosphorylated by choline kinase (CK); thereafter, phosphocholine condenses with CTP to form CDP-choline in a reaction catalysed by the CTP:phosphocholine cytidylyltransferase (CCT). CDP-choline, in turn, donates the phosphocholine moiety to diacylglycerol to form PtdCho by mediation of the DAG:CDP-choline transferase (CPT). The biosynthetic pathway is generally controlled by CCT, which is considered the rate-limiting enzyme [2]. CCT is an amphitropic protein, whose enzymatic activity is regulated by its association with membranes [3,4]. It corresponds to a class of amphitropic protein that contains no binding pockets for lipid monomers, but responds to changes in membrane physical properties, such as changes in surface density or lipid composition [4]. Four isoforms of CCT, CCTα, CCTβ1, CCT β2 and CCT β3, have been characterised. The beta isoforms are splicing variants of the same gene, whereas the alpha isoform is encoded by a separate gene [5]. The four isoforms are similar in their catalytic and membrane binding domains and are all regulated by lipids [5]. The mammalian CCT proteins are divided in four functional domains. The N-terminal domain of CCTα contains a nuclear localisation signal (NLS), whereas that of the CCTβ does not. A careful evaluation of CCT localisation in several cell types has demonstrated that CCTα is found associated with membranes in the cytoplasm, but that most of it is in the nucleus, as directed by the N-terminal nuclear localisation signal [6–8]. The soluble inactive nucleoplasmic pool of CCTα is considered a reservoir for a rapid response to extranuclear PtdCho synthesis [9]. In CHO, Hela and liver cells, nucleoplasmic CCTα translocates to the nuclear envelope for activation in response to several stimuli, including fatty acids [10], PtdCho degradation [11] and isoprenoids [12]. More recently, translocation of nuclear CCTα into intranuclear tubules of the nucleoplasmic reticulum has been found to promote its proliferation, triggered by fatty acids [13]. The mechanism of membrane CCTα insertion and activation has been extensively studied [14,15] but whether the nucleoplasmic pool of CCTα has any degree of organisation is unknown. It is now accepted that the mammalian nucleus is a highly structured and dynamic compartment that contains numerous morphologically well-defined structural units, including the nucleolus and several “nuclear bodies”, such as the Cajal body [16], and the promyelocytic leukaemia body [17]. Additionally, a large volume of nuclear space is occupied by a compartment commonly referred to as Biochimica et Biophysica Acta 1801 (2010) 1184–1194 Abbreviations: CCT, CTP:Phosphocholine Cytidylyltransferase; ER, endoplasmic reticulum; NR, nucleoplasmic reticulum; DIC, Differential interference contrast; TOTO-3, TOTO-3 iodide ⁎ Corresponding author. Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, 1er piso, (C1113AAD) Ciudad Autónoma de Buenos Aires, Argentina. Tel.: +5411 49648238; fax: +5411 49625457. E-mail address: speziale@ffyb.uba.ar (N.B. Sterin-Speziale). 1388-1981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2010.07.003 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip