Transbilayer movement of phosphatidylcholine in the mitochondrial outer membrane of Saccharomyces cerevisiae is rapid and bidirectional

Abstract

The process of transmembrane movement of phosphatidylcholine (PC) across the outer membrane of mitochondria was investigated in vitro in mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae. Phosphatidylcholine-transfer protein (PC-TP) was used to extract radiolabeled PC from OMV, with small unilamellar vesicles serving as acceptor system. Endogenously radiolabeled PC synthesized either via the CDP-choline pathway or via methylation of phosphatidylethanolamine can be extracted completely from the OMV with a t 1/2 of 1 min or less at 30°C. The size of the pool of PC in OMV available for exchange by PC-TP is not affected by pretreatment of the OMV with proteinase K or sulfhydryl reagents. In the reverse experiment where radiolabeled PC was introduced into the OMV, similar characteristics for the exchange were found. The accessibility of labeled PC to externally added phospholipase A 2 was used as a measure for its transmembrane distribution. It was found that PC is not exclusively located in the outer leaflet of the OMV. Only 30–35% can be degraded in intact OMV by phospholipase A 2, irrespective of whether the PC is introduced by PC-TP or endogenously synthesized via either of the pathways of biosynthesis. The results demonstrate the occurrence of rapid bidirectional transbilayer movement of both endogenous and in vitro introduced PC in OMV. Furthermore, there appears to be no preference for mitochondrial import of PC synthesized by either of the pathways in vivo.