Regulation of phosphatidylcholine synthesis by cellular metabolites and enzyme localization.
Date
2004
Authors
Lagace, Thomas Alfred.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
CTP:phosphocholine cytidylyltransferase (CCT)alpha is a ubiquitously expressed nuclear-localized enzyme in mammalian cells that catalyzes the rate-limiting and regulated reaction in the CDP-choline pathway for the synthesis of the major membrane phospholipid phosphatidylcholine (PtdCho). Soluble CCTalpha is activated upon binding to membranes via an amphipathic alpha-helical domain. To better understand the regulation of PtdCho synthesis and its integration with other cellular processes we examined the control of CCTalpha activity by membrane translocation and resultant effects on PtdCho synthesis and cell viability. In Chinese hamster ovary (CHO) cells with increased activity of sterol regulatory element binding proteins (SREBPs), PtdCho synthesis was upregulated coordinately with cholesterol synthesis due to CCTalpha translocation to the nuclear envelope (NE) and activation. This process was dependent on SREBP-mediated fatty acid synthesis. The cholesterol intermediate farnesol also induced CCTalpha activation in CHO cells. Farnesol-stimulated PtdCho synthesis was transient followed by profound inhibition. This coincided with CCTalpha nuclear export and apoptosis, which was prevented by supplementation of cells with CDP-choline pathway substrate (fatty acids or DAG). During apoptosis, CCTalpha was proteolyzed by caspase activity resulting in removal of its nuclear localization signal, implying an essential role of CCTalpha nuclear localization. Sites of intranuclear CCTalpha membrane binding in response to the fatty acid activator oleate were examined. Under these conditions, CCTalpha associated with and caused an increase in tubules of the nucleoplasmic reticulum (NR) continuous with the NE and containing cytoplasm and endoplasmic reticulum components. Electron microscopy analysis identified membrane arrays associated with oleate-induced tubules suggesting the NR could be a site of PtdCho synthesis. Membrane binding ability of CCTalpha was necessary for oleate-induced nuclear tubule formation and CCTalpha directly tubulated liposomes in vitro. These results support that regulation of CCTalpha by membrane binding and nuclear localization coordinates PtdCho and cholesterol synthesis and affects the cellular processes of apoptosis and NR formation.
Thesis (Ph.D.)--Dalhousie University (Canada), 2004.
Thesis (Ph.D.)--Dalhousie University (Canada), 2004.
Keywords
Biology, Molecular.