CTP:Phosphocholine Cytidylyltransferase α Is a Cytosolic Protein in Pulmonary Epithelial Cells and Tissues

Abstract

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in de novo synthesis of phosphatidylcholine (PC). The lung requires a steady synthesis of PC for lung surfactant of which disaturated PC is the essential active agent. Surfactant synthesis occurs in alveolar type II cells. Studies with non-pulmonary cells have suggested that CCT is both a nuclear and cytoplasmic protein. The unusual requirements of the lung for PC synthesis and, therefore, CCT activity suggest a unique mechanism of regulation and possibly localization of CCT. The localization of CCTα in lung epithelial cells and, of greater consequence, lung tissues are yet unknown. Three isoforms of CCT have been identified. Herein we investigated the localization of the ubiquitously expressed CCTα isoform. To ascertain CCTα localization in lungs and lung-related epithelial cells, we employed a number of localization methods. Immunogold electron microscopy using polyclonal antibodies raised to either the carboxyl terminus, catalytic domain, or amino terminus of CCTα localized CCTα mostly to the exterior plasma membrane or regions of the endoplasmic reticulum (ER) in both A549 and MLE-15 epithelial lung cell lines and primary cultures of fetal rat lung epithelial cells. In contrast to other studies, little or no nuclear labeling was observed. Indirect immunofluorescence of these cells with anti-CCTα antibodies resulted in a similar distribution. Indirect visualization of both hemagglutinin- and FLAG-tagged CCTα as well as direct visualization of enhanced green fluorescence protein-CCTα fusion protein corroborated a cytoplasmic localization of CCTα in pulmonary cells. Moreover, analysis of lung tissue from fetal and adult mouse by either immunogold electron microscopy or indirect immunofluorescence yielded a strong cytoplasmic CCTα signal with virtually no nuclear localization in epithelial cells lining the airways. The cytoplasmic localization of CCTα in type II cells was further substantiated with transgenic mice overexpressing FLAG-tagged CCTα using the lung-specific human surfactant protein C (SP-C) promoter. We conclude that CCTα does not localize to the nucleus in pulmonary tissues, and, therefore, nuclear localization of CCTα is not a universal event.