Functions of membrane binding domain of CTP:phosphocholine cytidylyltransferase in alveolar type II cells.

CTP:phosphocholine cytidylyltransferase (CCT alpha) 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-CCT alpha constructs were generated to evaluate the subcellular distribution in A549 cells. The M domain targeted CCT alpha 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 VEEKS267-277 motif of the M domain resulted in a nuclear appearance and indented nuclei. Membrane binding of CCT alpha 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 CCT alpha, we visualized the protein in the absence and presence of lipids by transmission electron microscopy. These studies revealed that CCT alpha forms a dimer-like complex that condenses upon binding to lipid vesicles, but not lipid monolayers. The influence of the M domain on CCT alpha activity was assessed in transgenic mice overexpressing the N-terminal catalytic domain (CCT alpha(1-239)), N-terminal catalytic plus M domain (CCT alpha(1-290)), or full-length CCT alpha(1-367) in fetal type II cells by using the surfactant protein C promoter. Only overexpression of CCT alpha(1-367) increased surfactant phosphatidylcholine synthesis. Thus, the M domain influences membrane binding, cellular distribution, and topology of CCTa, but the domain alone is not sufficient to confer CCT activity in alveolar type II cells in vivo.