Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity

Abstract

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.

Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants

“…Iodide Quenching of Fl-PL in ABCA Proteoliposomes-To assess the steady-state distribution of the Fl-PL across the membranes of proteoliposomes, collisional quenching of fluorescent lipid probes was performed with potassium iodide (KI) (38). ABCA protein was reconstituted using Buffer C but with 250 mM NaCl and devoid of sucrose.…”

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

“…The t 1/2 of this translocation is in the order of hours to days (Rothman & Kennedy 1977, Gummadi & Kumar 2005. However, in biogenic membranes, t 1/2 of Flippase activity in epididymal sperm membranes PL translocation was calculated to be in the order of few minutes with reports indicating their presence in ER, Golgi complex and chloroplasts (Bishop & Bell 1985, Backer & Dawidowicz 1987, Hrafnsdóttir et al 1997, Menon et al 2000, Gummadi & Menon 2002, Kubelt et al 2002, Chang et al 2004, Rajasekharan & Gummadi 2011). These biogenic membrane flippases are different from ATP-dependent flippases, which are involved in the maintenance of membrane asymmetry at PM (Holthuis & Levine 2005, Pomorski & Menon 2006.…”

“…reconstitution buffer (10 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, and 1% (w/v) Triton X-100) respectively. The sample was mixed and incubated at 4 8C with end-over-end rocking for 1 h. It was then ultracentrifuged in a MLA 130 rotor at 175 000 g for 30 min at 4 8C (Rajasekharan & Gummadi 2011). The supernatant was carefully removed and used directly for reconstitution at a protein concentration of w3 mg/ml.…”

“…This experiment was performed to verify the reconstitution of membrane proteins from TE as described previously (Rigaud et al 1988, Rajasekharan & Gummadi 2011. Briefly, 1 ml of the reconstituted proteoliposomes was layered on a series of sucrose (w/w) (20, 10, 5, and 2.5%) and centrifuged at 100 000 g in a swing-out rotor (SW 41Ti) for 1 h. Fractions were collected from different sucrose interfaces and dialyzed against assay buffer for 24 h with three to four buffer changes to remove sucrose from the fractions.…”

“…The assay was performed using Perkin Elmer LS-55 Fluorescence Spectrophotometer as reported previously (Sahu & Gummadi 2008, Rajasekharan & Gummadi 2011. A total of 50 ml of labeled liposomes and proteoliposomes were added to 1.95 ml of assay buffer in a fluorescence cuvette.…”