Abstract
One of the steps in the import of phosphatidylcholine (PC) in mitochondria is transmembrane movement across the outer membrane. This process was investigated in vitro using isolated mitochondrial outer membrane vesicles (OMV) from rat liver. 14C-Labeled PC was introduced into the OMV from small unilamellar vesicles by a PC-specific transfer protein (PCTP). The membrane topology of the newly introduced PC was determined from its accessibility to phospholipase A2. Under conditions where the OMV stay intact, externally added phospholipase A2 is able to hydrolyze up to 50% of both the introduced [14C]PC and the endogenous PC. Pool size calculations showed that close to 100% of the PC in the OMV can be exchanged by PCTP. A back-exchange experiment revealed that the introduction of the labeled PC is reversible. The results demonstrate that newly introduced PC molecules readily equilibrate over both leaflets of the OMV membrane. The kinetics of the PCTP-mediated exchange process indicate that the t1/2 of the transmembrane movement at 30 degrees C is 2 min or less.
Highlights
Phosphatidylcholine (PC)1 is the major constituent phospholipid in both the inner and the outer membrane of mammalian mitochondria [1]
Localization of [14C]DOPC in outer membrane vesicles (OMV) by Treatment with phospholipase A2 (PLA2)—14C-Labeled DOPC was introduced into isolated mitochondrial OMV from donor vesicles by the action of the Phosphatidylcholine-specific transfer protein (PCTP)
This protein catalyzes a one-to-one PC exchange resulting in a replacement of the endogenous PC in the acceptor membrane with PC from the donor vesicles, i.e. without changing the PC content of the membranes [23,24,25]. [14C]DOPC was efficiently transferred from donor small unilamellar vesicles (SUV) to acceptor OMV under the conditions used, an incubation for 20 min at 30 °C with equal amounts of PC present in the populations of donor and acceptor vesicles (PCdonor/PCacceptor molar ratio approximately 1), and in the presence of PCTP
Summary
Phosphatidylcholine (PC)1 is the major constituent phospholipid in both the inner and the outer membrane of mammalian mitochondria [1]. In back-exchange experiments, OMV with [14C]DOPC introduced as described above were incubated at 0.25 mg/ml with unlabeled SUV consisting of DOPC/DOPA (95:5, mol/mol) at a concentration of 1.5 mM based on phosphorus, in the presence of 18 g/ml PCTP at 30 °C for the periods of time indicated.
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