Gehrig Karsten, Morton Craig C, Ridgway Neale D
Department of Pediatrics, and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia, Canada.
J Lipid Res. 2009 May;50(5):966-76. doi: 10.1194/jlr.M800632-JLR200. Epub 2008 Dec 20.
phosphocholine cytidylyltransferase alpha (CCTalpha), the rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis, is activated by translocation to nuclear membranes. However, CCTalpha 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 CCTalpha 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 CCTalpha-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 nuclear-localized 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 lipid-dependent activation to the nuclear/cytoplasmic distribution of CCTalpha.
磷酸胆碱胞苷转移酶α（CCTα）是磷脂酰胆碱（PtdCho）合成的CDP-胆碱途径中的限速酶，通过转位至核膜而被激活。然而，在PtdCho合成能力增强的细胞以及急性激活后，CCTα位于细胞质中，这表明核输出与激活有关。本研究的目的是确定CCTα的哪些结构域参与了对脂质激活剂法尼醇（FOH）和油酸的核输出过程。对在CCTα缺陷的CHO58细胞中表达的CCT-绿色荧光蛋白（GFP）突变体进行成像显示，FOH介导的转位至核膜以及输出至细胞质需要膜结合两亲性螺旋（结构域M）。模拟CCT磷酸化（P）结构域组成型磷酸化的突变会减少核输出。然而，单独的结构域M就足以促进在经FOH或油酸处理的CHO58细胞中转位至核膜以及输出一种核定位的GFP构建体。在脂质介质急性激活的情况下，CCT-GFP突变体的核输出与体外活性相关，但与PtdCho合成无关。本研究描述了一种依赖于两亲性螺旋膜相互作用的核输出途径，从而将脂质依赖性激活与CCTα的核/细胞质分布联系起来。
