Abstract
Phosphatidylcholine (PC) translocation was studied in rat liver canalicular plasma membrane vesicles using a fluorescent PC analogue that permitted the quantitation of asymmetric PC distribution in the outer and inner leaflet of the vesicles. PC translocation to the outer leaflet of the canalicular membrane was stimulated by ATP and an ATP-regenerating system in a time and temperature-dependent manner resulting in 200 pmol PC translocated/mg protein per 30 min. A non-hydrolyzable ATP analogue did not support translocation. Translocating activity was observed with PC but not with phosphatidylethanolamine and was specific for inside-out oriented canalicular membrane vesicles. Addition of taurocholate (10 microM), a micelle-forming bile acid, enhanced ATP-dependent PC translocation 1.5 +/- 0.1-fold, whereas addition of taurodehydrocholate (10 microM), a non-micelle-forming bile acid, did not. These results indicate the presence of an ATP-dependent transporter that "flips" phosphatidylcholine from the inner to the outer leaflet of the rat bile canalicular plasma membrane from where it can become associated with bile acids in the canalicular lumen, thereby enhancing ATP-dependent flipping activity. Several lines of evidence suggest that the transporter is Mdr2 P-glycoprotein.
Highlights
Phosphatidylcholine (PC) translocation was studied in rat liver canalicular plasma membrane vesicles using a fluorescent PC analogue that permitted the quantitation of asymmetric PC distribution in the outer and inner leaflet of the vesicles
This was further supported by the finding that in canalicular membrane vesicles (CMV), incubated with ATP, 0.05% Triton was sufficient to render Cs-NBD-PC molecules associated with the inner leaflet accessible to dithionite (Fig. 5C), whereas, in ATP and 10 ~ L MTC (Fig. 5D), 0.1%of Triton was necessary to render the CG-NBD-PCmolecules accessible to dithionite reduction (n = 3).These results suggest that some Cs-NBD-PCmolecules were present in micelles or aggregates, which required a higher detergent concentration for complete solubilization
Because preparation of fluorescently labeled CMV and the phospholipid translocation assay were adapted from studies by Ruetz and Gros in yeast [21], it was first necessary to validate these methods
Summary
Ented vesicles were separated from right-side-out oriented vesicles by antibody-induced density perturbation. Donor liposomes that were labeled with Cs-NBD-PC only in the inner leaflet were used. These "only inside-labeled donor liposomes" were prepared by incubating donor liposomes for 30 min with 40 mM dithionite and subsequent dialysis at 4°Cfor 16 h against buffer A. Transfer was initiated by mixing CMV with donor liposomes at a concentration of 17 nmol Cs-NBD-PC/mg protein. After incubating the mixture for 30 min at 4"C,free donor liposomes were separated from NBD-labeled CMV by centrifugation of the mixture at 100,000g for 30 min in a swinging bucket rotor through 16% sucrose (16% sucrose in 10 mM TrisHEPES, pH 7.4, 0.2 mM CaC12). When Student’s t-test was used to determine statistical significance, P values are given
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