Abstract
Phosphatidylinositol transfer protein (PI-TP) is one of a group of proteins that are cytosolic proteins characterized by their ability to facilitate the transfer of phospholipids between membranes in vitro. It has recently been demonstrated that yeast PI-TP (SEC14p) inhibited Golgi membrane cholinephosphate cytidylyltransferase (CYTase) in vitro andin vivo (Skinner HB, et al. Proc. Natl. Acad. Sci. 1995, 92:112-116). We hypothesized that PI-TP regulates CYTase in rat alveolar type II cells (TII). TII cells were freshly isolated from adult rat lung by Nycodenz gradient centrifugation and cytosol (cyt) protein obtained by digitonin release and microsome (mc) fractions obtained by differential centrifugation. Fractions (50-100μg) were incubated with 0.5μg rrPI-TP(gift G.M. Helmkamp, Jr., Kansas, KS) or buffer in total volume of 1 ml at 37°C for 5 to 30 min. Two forms of rrPI-TP were used - one bound to phosphatidylglycerol (PG) and one bound to phosphatidylcholine (PC). Cytosolic CYTase was stimulated 2.1- and 7.9-fold by PG-rrPI-TP and PC-rrPI-TP, respectively. There was 1.2-fold greater stimulation of mc CYTase by PC-rrPI-TP than PG-rrPI-TP. Co-incubation with PI/PC (90:10, mol:mol) vesicles or chlorpromazine, a PI-TP inhibitor, abolished the stimulatory effect. Immunoprecipitation of PI-TP in TII cyt with rabbit anti-PI-TP antibody decreased CYTase activity 47%. These results suggest that 1) PI-TP stimulates TII CYTase; 2) the interaction of PI-TP with CYTase is dependent, in part, on the bound phospholipid; 3) lipid vesicles may compete with mc membrane for PI-TP. PI-TP may be an important regulator of CYTase activation and/or membrane binding. Funded by NIH R29 HL44853.
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