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. 2011 Dec 28;17(1):136–152. doi: 10.2478/s11658-011-0042-8

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

Archita Rajasekharan 1, Sathyanarayana N Gummadi 1,✉
PMCID: PMC6275754  PMID: 22207336

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 alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.

Key words: Biogenic membrane flippase, Cholesterol, Endoplasmic reticulum, Flip flop

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Abbreviations used

DTT

dithiothreitol

ePC

egg phosphatidylcholine

ER

endoplasmic reticulum

HEPES

N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid

HLT1

first half-life time

HLT2

second half-life time

MOPS

3-(N-morpholino)propanesulfonic acid

NBD-PC

1-oleoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)aminododecanoyl}-sn-glycero-3-phosphocholine

PC

phosphatidylcholine

PL

phospholipid

PM

plasma membrane

PMSF

phenylmethanesulfonylfluoride

SWER

salt-washed endoplasmic reticulum

TE

Triton X-100 extract

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