A novel assay to measure scrambling of natural phospholipids in reconstituted proteoliposomes

Abstract

Scramblases transport phospholipids between the leaflets of a membrane bilayer in an ATP‐independent and bidirectional way to facilitate membrane growth and maintain lipid homeostasis. Members of two protein families, Class A G protein‐coupled receptors and the TMEM16 family of ion channels, have been identified to act as scramblases. However, the mechanisms by which they translocate phospholipids are still poorly understood. Synthetic glycerophospholipid analogs carrying fluorescent nitrobenzoxadiazole (NBD) groups, such as NBD‐phosphatidylcholine (NBD‐PC), have proven useful to identify and characterize phospholipid scramblases in membrane preparations and reconstituted proteoliposomes.We now have established a novel biochemical assay to measure the activity of scramblase proteins using radiolabeled natural phosphatidylinositol ([3H]PI) as probe. As sources of scramblase activity we used crude extracts from yeast containing ER membrane proteins, and also purified bovine opsin. We reconstituted symmetric large unilamellar liposomes containing trace amounts of [3H]PI. Addition of PI‐specific phospholipase C (PI‐PLC) resulted in hydrolysis of 46–52% of [3H]PI, i.e. the portion present in the outer leaflet of the bilayer. The time to reach maximal [3H]PI hydrolysis depended on the amount of PI‐PLC used. In contrast, PI‐PLC treatment of [3H]PI‐containing liposomes reconstituted with yeast extract containing scramblase activity or purified opsin resulted in protein‐dependent hydrolysis of 60–80% of [3H]PI, indicating that the [3H]PI located in the inner leaflet of the proteoliposomes became accessible to cleavage by PI‐PLC during the course of the experiment.To allow direct comparison of opsin's ability to scramble [3H]PI and NBD‐PC, we then prepared opsin proteoliposomes containing both [3H]PI and NBD‐PC and measured scrambling using PI‐PLC and dithionite, respectively, as probes. The results showed that opsin readily scrambled both NBD‐PC and [3H]PI, albeit with different characteristics. The results of these experiments, as well as experiments that directly compare opsin‐mediated scrambling of NBD‐PI and [3H]PI will be presented.apoproteinSupport or Funding InformationThis research is supported by the Velux StiftungThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.