TRANSMEMBRANE MOVEMENT OF PHOSPHATIDYLCHOLINE IN MITOCHONDRIAL OUTER-MEMBRANE VESICLES

One of the steps in the import of phosphatidylcholine (PC) in mitochon dria is transmembrane movement across the outer membrane, This process was investigated in vitro using isolated mitochondrial outer membrane vesicles (OMV) from rat liver. C-14-Labeled PC was introduced into th e OMV from small unilamellar vesicles by a PC-specific transfer protei n (PCTP). The membrane topology of the newly introduced PC was determi ned from its accessibility to phospholipase A(2). Under conditions whe re the OMV stay intact, externally added phospholipase A(2) is able to hydrolyze up to 50% of both the introduced [C-14]PC and the endogenou s PC. Pool size calculations showed that close to 100% of the PC in th e OMV can be exchanged by PCTP. A back-exchange experiment revealed th at the introduction of the labeled PC is reversible. The results demon strate that newly introduced PC molecules readily equilibrate over bot h leaflets of the OMV membrane. The kinetics of the PCTP-mediated exch ange process indicate that the t(1/2) of the transmembrane movement at 30 degrees C is 2 min or less.