Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

Abstract

Abstract:Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

“…The scrambling activity was lower when Chol was present in the membrane (Fig. 6, C and D), showing that the Chol ordering effect hampers the transbilayer distribution of lipids, in agreement with other studies (44)(45)(46).…”

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis

“…We hypothesize that defective cholesterol removal mechanism might have led to the retention of cholesterol, which would have subsequently inhibited the flippases that modulate the sperm membranes. It has been reported that cholesterol inhibits flippase activity even at very low concentrations such as 1 mol% (Rajasekharan & Gummadi 2012). To check this effect, we reconstituted the whole cell and mitochondria with 15 mol% cholesterol and our results confirmed that the presence of cholesterol inhibited flippase activity only in whole cells and not in mitochondria, which is consistent with earlier reports that have shown that spermatozoa continuously modulate membrane cholesterol content during their transport and maturation (Cross 1998, Apel-Paz et al 2003.…”