The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha.
CTP:phosphocholine cytidylyltransferase a (CCTa), the rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis, is activated by translocation to nuclear membranes. However, CCTa is cytoplasmic in cells with increased capacity for PtdCho synthesis and following acute activation, suggesting that nuclear export is linked to activation. The objective of this study was to identify which CCTa domains were involved in nuclear export in response to the lipid activators farnesol (FOH) and oleate. Imaging of CCT-green fluorescent protein (GFP) mutants expressed in CCTa-deficient CHO58 cells showed that FOH-mediated translocation to nuclear membranes and export to the cytoplasm required the membrane binding amphipathic helix (domain M). Nuclear export was reduced by a mutation that mimics constitutive phosphorylation of the CCT phosphorylation (P) domain. However, domain M alone was sufficient to promote translocation to the nuclear envelope and export of a nuclearlocalized GFP construct in FOH-or oleate-treated CHO58 cells. In the context of acute activation with lipid mediators, nuclear export of CCT-GFP mutants correlated with in vitro activity but not PtdCho synthesis. This study describes a nuclear export pathway that is dependent on membrane interaction of an amphipathic helix, thus linking lipiddependent activation to the nuclear/cytoplasmic distribution of CCTa.-Gehrig, K., C. C. Morton, and N. D. Ridgway. Nuclear export of the rate-limiting enzyme in phosphatidylcholine synthesis is mediated by its membrane binding domain. J. Lipid Res. 2009. 50: 966-976.
Phosphatidylcholine (PtdCho), a zwitterionic glycerophospholipid that comprises 40-60% of eukaryotic membrane mass, is an essential component of lung surfactant, bile, and lipoproteins, and a source for signaling molecules such as diacylglycerol (DAG) and phosphatidic acid
In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.