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

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.

Abbreviations

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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Correspondence to Sathyanarayana N. Gummadi.

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Rajasekharan, A., Gummadi, S.N. Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels. Cell Mol Biol Lett 17, 136–152 (2012). https://doi.org/10.2478/s11658-011-0042-8

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Key words

  • Biogenic membrane flippase
  • Cholesterol
  • Endoplasmic reticulum
  • Flip flop