Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter
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Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter. / Mathiassen, Patricia P. M.; Menon, Anant K.; Pomorski, Thomas Guenther.
I: Scientific Reports, Bind 11, Nr. 1, 14364, 2021.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter
AU - Mathiassen, Patricia P. M.
AU - Menon, Anant K.
AU - Pomorski, Thomas Guenther
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - MEMBRANE-PROTEINS
KW - FLIP-FLOP
KW - MECHANISMS
KW - PHOSPHATIDYLSERINE
KW - ELECTROFORMATION
KW - ORGANIZATION
KW - MIXTURES
KW - DYNAMICS
KW - EXPOSURE
KW - MOBILITY
U2 - 10.1038/s41598-021-93664-0
DO - 10.1038/s41598-021-93664-0
M3 - Journal article
C2 - 34257324
VL - 11
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 14364
ER -
ID: 275434626