Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

Abstract

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.

In vitro and in vivo assay of the ER lipid scramblase TMEM41B

“…In the presence of a scramblase (TMEM41B), which catalyzes the exchange of NBD-lipids between the leaflets, greater fluorescent quenching will be observed due to exposure of inner leaflet NBD to dithionite on the outer leaflet. Similar studies have been carried out previously on related scramblases such as Triton-extracted ER scramblases, opsin, TMEM16, ATG9 and VMP1 ( Brunner et al., 2014 ; Ghanbarpour et al., 2021 ; Hrafnsdóttir and Menon, 2000 ; Mathiassen et al., 2021 ; Matoba et al., 2020 ; Menon et al., 2011 ; Suzuki et al., 2013 ; Vehring et al., 2007 ).…”

“…Here we describe the reconstitution of TMEM41B into liposomes to characterize its scramblase activity using a fluorescence liposome-based in vitro scramblase assay ( Brunner et al., 2014 ; Ghanbarpour et al., 2021 ; Hrafnsdóttir and Menon, 2000 ; Marek and Günther-Pomorski, 2016 ; Mathiassen et al., 2021 ; Matoba et al., 2020 ; Menon et al., 2011 ; Ploier and Menon, 2016 ; Vehring et al., 2007 ). Trace amounts of 7-nitro-2-1,3-benzoxadiazol-4-yl acyl chain-labeled phosphatidylcholine (NBD-PC) adapts a ∼50% distribution between the inner and the outer leaflet of a symmetric liposome bilayer.…”

“…In this protocol, we demonstrated phospholipids scramblase activity of the recombinant human FLAG-TMEM41B by a fluorescence liposome-based in vitro scramblase assay ( Brunner et al., 2014 ; Ghanbarpour et al., 2021 ; Hrafnsdóttir and Menon, 2000 ; Marek and Günther-Pomorski, 2016 ; Mathiassen et al., 2021 ; Matoba et al., 2020 ; Menon et al., 2011 ; Ploier and Menon, 2016 ; Vehring et al., 2007 ). We showed that the phospholipids scramblase activity of TMEM41B is concentration dependent.…”

“…We suggest to set a suitable period of time to monitoring the sample and reduce the number of samples to reduce inspection interval (because the more samples you have, the longer inspection interval time you need). It is also recommended to determine the presence of Triton X-100 (absorbance at 275 nm) after reconstitution ( Hrafnsdóttir and Menon, 2000 ) to exclude dithionite leakage over the liposome bilayer over time ( Mathiassen et al., 2021 ). Decreasing fluorescence traces may also be due to the use of a plate reader, as the vesicles would sediment over time in the absence of stirring.…”

“…Decreasing fluorescence traces may also be due to the use of a plate reader, as the vesicles would sediment over time in the absence of stirring. A suggestion could be to use a fluorometer cuvette with stirring instead of the 96-well plate, as described in ( Marek and Günther-Pomorski, 2016 ; Mathiassen et al., 2021 ; Ploier and Menon, 2016 ).…”