Functional studies on the phosphatidychloride transfer protein

Abstract

The phosphatidylcholine transfer protein (PC-TP) has been studied for over 30 years now. Despite extensive research concerning the biochemical, biophysical and structural properties of PC-TP, the function of this protein is still elusive. We have studied in vitro the folding and the mechanism of PC extraction from membranes. In vivo, we have assessed previously postulated and novel physiological functions of PC-TP by localization studies in various cell lines and by examining lipid metabolism in cells overexpressing PC-TP and Pc-tp null mice. PC proved to be indispensable for the folding of PC-TP as shown by refolding from inclusion bodies. By incubation of PC-TP with microsomes it was confirmed that PC-TP has the highest affinity for PC species with a C16:0 on the sn-1 and a polyunsaturated fatty acid (PUFA) on the sn-2 position. Using electrospray time-of-flight mass spectrometry we established that these complexes are less stable than the PC-TP/C18:0/PUFA-PC complexes. We propose that enhanced take-up and release rates eventually accelerate the transfer of especially C16:0/PUFA-PC. PC-TP is evenly distributed throughout the cytoplasm of foetal bovine heart endothelial cells, human umbilical vein endothelial cells, HepG2 cells and NIH3T3 mouse fibroblasts. As measured by fluorescence recovery after bleaching, PC-TP was found to be highly mobile throughout the cell. After the addition of peroxisome proliferators, PC-TP relocated to mitochondria in endothelial cells within 5 min. This process is probably mediated by phosphorylation of Ser110 present in the sole putative PKC phosphorylation site of PC-TP. Pc-tp null mice were generated to study the a number of previously proposed functions for the PC-TP, such as a role in bile formation, lung surfactant production and the facilitation of enzymatic reactions involving PC synthesis or breakdown. Remarkably, these mice were normal and had no defect in any of the postulated PC-TP functions analyzed. However, the body weight of the Pc-tp (-/-) mice was 15% higher than that of Pc-tp (+/+) mice after six months on a regular diet. By challenging these mice with diets containing clofibrate and phytol for a period of three weeks, we established a clear connection between PC-TP and these peroxisomal proliferators. PC-TP levels increased up to 7.0-fold in Pc-tp (+/+) mice, leading to a reduction of total triglyceride and phospholipid levels in blood plasma. These effects were absent in the Pc-tp (-/-) mice. It is clear that the physiological function of PC-TP is related to the effects of peroxisome proliferators on lipid metabolism in the body. Since PC-TP relocates to mitochondria, it is inevitable to focus our attention to this organelle. We think that the main target of PC-TP is the â-oxidation of fatty acids. At an influx of an excess of fatty acids, PC-TP relocates to mitochondria and stimulates the â-oxidation. At the same time, PC-TP is upregulated via its PPAR-alpha responsive element. The next time an excess of fatty acids is presented to the cell, the cell can cope more effectively with these lipids.