A cholesterol switch controls phospholipid scrambling by G protein–coupled receptors

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG

“…Thus, the presence or absence of Rft1 does not affect the outcome of the M5- DLO scramblase assay. We note that a larger proportion of proteoliposomes contain phospholipid scramblase activity compared with M5-DLO scramblase activity, ∼55% versus ∼30%, respectively (calculated as described (46)), consistent with the greater abundance of phospholipid scramblase(s) in the TE as reported previously (5).…”

“…The samples were removed from the BioBeads and centrifuged in a Beckman TLA100.2 rotor at 75,000 rpm (∼250,000 x g max )at 4°C for 1 h. The resulting membrane pellets were each resuspended in 200 μl MMC buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 3 mM MgCl 2 ,1 mM MnCl 2 ,1 mM CaCl 2 ). Aliquots of the resuspended pellets were taken for dynamic light scattering and phospholipid scramblase activity assays as described (45), and M5-DLO scramblase assay according to a standard protocol (21).…”

“…3C)). These numbers indicate that, at the protein/phospholipid ratio used for reconstitution, approximately 55% of the vesicles contain a phospholipid scramblase (calculated as described (45)). Furthermore, the identical fluorescence reduction traces obtained for proteoliposomes generated from the treated and mock-treated samples indicate that these vesicles are similarly reconstituted.…”

Imaging single particle profiler to study nanoscale bioparticles using conventional confocal microscopy

“…However, standard deviation (designated as “σ” which is the full width at half maxima) for POPC/Chol is higher than for the other two, displaying higher heterogeneity of membrane fluidity within this sample. This is due to imperfect mixing of lipids in multi-component systems [13] , hence σ in SPP measurements can be used to investigate the sample heterogeneity [14] .…”

“…Cholesterol encapsulation by MBCD is widely used to remove cholesterol from native and artificial lipid bilayers, which is a standard procedure when the role of cholesterol in various biological processes is in question [16,[18][19][20] . Interestingly, it is also possible to use MBCD as an intermediary to deliver and distribute cholesterol homogeneously among artificial liposomes [14] . MBCD interaction with membranes is complex especially when there are multiple different lipid environments present.…”