Flippase Activity in Proteoliposomes Reconstituted with Spinacea oleracea Endoplasmic Reticulum Membrane Proteins: Evidence of Biogenic Membrane Flippase in Plants

Abstract

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

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

“…This implies that second phase quenching is slower because of transbilayer movement of PLs from the inner leaflet to the outer leaflet. The results obtained in this study are consistent with the half-life time reported earlier by several researchers [10][11][12].…”

“…The microsomal membrane fraction was prepared as described previously [12,13]. Briefly, 60 g of fresh leaves were homogenized in 60 ml of buffer A [MOPS-BTP buffer (pH 7.5) and 0.33 M sucrose supplemented with 5 mM EDTA, 5 mM KCl, 5 mM DTT, 0.5 mM PMSF, 1 mM MgCl 2 and 0.2% (w/v) BSA].…”

“…The assay was performed using a Perkin Elmer LS-5S Fluorescence spectrophotometer as described earlier [12]. A total of 20 μl of NBD-PC labeled liposomes and proteoliposomes was added to 1.98 ml of 10 mM HEPES/NaOH pH 7.5 and 100 mM NaCl in a fluorescence cuvette.…”

“…A sharp decrease in fluorescence to ~ 50-55% was observed upon dithionite addition, followed by a slow decrease in fluorescence. The sharp decrease in fluorescence is due to quenching of labeled lipids on the outer leaflet, whereas the slower decrease in fluorescence is due to flipping of inner labeled PLs to the outer leaflet, which subsequently gets quenched by dithionite [12,24]. However, upon addition of 0.1% (w/v) Triton X-100 to liposomes, ~ 100% quenching was observed, indicating that the membrane permeability was disturbed, leaving all the PLs accessible to dithionite for quenching (data not shown).…”

“…However, biogenic or self-synthesizing membranes are equipped with a special class of protein translocators called biogenic membrane flippases, which are involved in rapid flip-flop of PLs (irrespective of PL head group) and are metabolic energy independent, thereby differing from conventional ATP-dependent flippases which are localized at non-biogenic membranes such as plasma membrane (PM) [9]. Biogenic membrane flippase activity has been shown in bacterial cytoplasmic membranes, and ER of animals and plants [10][11][12]. We hypothesize that cholesterol affects the rapid flip-flop of PLs across the bilayer.…”