Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, ON, M5G 1X8 Canada.
Am J Respir Cell Mol Biol. 2010 Jul;43(1):74-87. doi: 10.1165/rcmb.2009-0231OC. Epub 2009 Aug 14.
phosphocholine cytidylyltransferase (CCTalpha) plays a key role in the biosynthesis of surfactant phosphatidylcholine. In this study, we investigated the role of its membrane-binding (M) domain in modulating its structure, function, and cellular distribution. Multiple enhanced green fluorescent protein-CCTalpha constructs were generated to evaluate the subcellular distribution in A549 cells. The M domain targeted CCTalpha to the perinuclear (membrane-rich) region. Microinjections with glutathione-S-transferase fusion protein containing the M domain corroborated the perinuclear targeting. Deletion of the M domain or substitutions of the hydrophobic residues with arginine/serine in the VEEKS(267-277) motif of the M domain resulted in a nuclear appearance and indented nuclei. Membrane binding of CCTalpha decreased gradually as the number of positively charged arginine residues increased in the VEEKS motif. To identify whether membrane-protein interactions cause structural alterations in CCTalpha, we visualized the protein in the absence and presence of lipids by transmission electron microscopy. These studies revealed that CCTalpha forms a dimer-like complex that condenses upon binding to lipid vesicles, but not lipid monolayers. The influence of the M domain on CCTalpha activity was assessed in transgenic mice overexpressing the N-terminal catalytic domain (CCTalpha(1-239)), N-terminal catalytic plus M domain (CCTalpha(1-290)), or full-length CCTalpha(1-367) in fetal type II cells by using the surfactant protein C promoter. Only overexpression of CCTalpha(1-367) increased surfactant phosphatidylcholine synthesis. Thus, the M domain influences membrane binding, cellular distribution, and topology of CCTalpha, but the domain alone is not sufficient to confer CCT activity in alveolar type II cells in vivo.
磷酸胆碱胞苷酰转移酶（CCTalpha）在表面活性剂磷脂酰胆碱的生物合成中发挥关键作用。在这项研究中，我们研究了其膜结合（M）结构域在调节其结构、功能和细胞分布中的作用。生成了多个增强型绿色荧光蛋白-CCTalpha 构建体，以评估其在 A549 细胞中的亚细胞分布。M 结构域靶向 CCTalpha 到核周（富含膜）区域。用含有 M 结构域的谷胱甘肽 S-转移酶融合蛋白进行微注射证实了核周靶向。M 结构域中 VEEKS（267-277）基序的疏水残基缺失或用精氨酸/丝氨酸取代会导致核内出现和核凹陷。随着 M 结构域中带正电荷的精氨酸残基数量的增加，CCTalpha 的膜结合逐渐减少。为了确定膜蛋白相互作用是否导致 CCTalpha 的结构改变，我们通过透射电子显微镜在没有和存在脂质的情况下可视化该蛋白。这些研究表明，CCTalpha 形成二聚体样复合物，在与脂质囊泡结合时会发生凝聚，但与脂质单层不发生凝聚。通过使用表面活性蛋白 C 启动子，在胎肺 II 型细胞中转基因小鼠中过表达 N 端催化结构域（CCTalpha(1-239)）、N 端催化加 M 结构域（CCTalpha(1-290)）或全长 CCTalpha(1-367)，评估了 M 结构域对 CCTalpha 活性的影响。只有过表达 CCTalpha(1-367)增加了表面活性剂磷脂酰胆碱的合成。因此，M 结构域影响 CCTalpha 的膜结合、细胞分布和拓扑结构，但该结构域本身不足以在体内赋予肺泡 II 型细胞中的 CCT 活性。
