Biogenic membrane flippases.
We chose plant as model system and tried to understand the membrane biogenesis in plants and reported the first evidence for biogenic membrane flippases in plants. Biogenic membrane flippases in plant ER exhibited bidirectional flipping of PLs, independent of PL head group and the translocation is protein mediated as evident from trypsin treatment and protein modification experiments.
We also showed 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 and (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner.
Fig 4. Proteoliposomes reconstituted with envelope membrane (white) and thylakoids (black) fractions quenched with dithionite (control), NBD-PG proteoliposomes incubated with 5 mM GTP, ADP, ATP and 10 mg/ml trypsin quenched with dithionite
Other than plants, we also studied the membrane biogenesis (ATP independent flippases) and lipid traffic in spermatozoa. We hypothesized that spermatozoa might contain biogenic membrane flippases since immense change in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes.
In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP independent flip-flop movement of phosphatidylcholine, phosphatidylserine and phosphatidylglycerol. Half-life time of phosphatidylcholine flipping was found to be ~ 3.2 ± 1 min for whole sperm membrane, which otherwise would have taken ~ 11-12 h in the absence of protein.
To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP independent flippase activity. Interestingly, mitochondrial membranes showed flip-flop but were specific for phosphatidylcholine with half-life time of ~ 5 ± 2 min.
Our results also suggest that spermatozoa have different populations of flippases and their localization within the cellular compartments depending on the type of phospholipid synthesis. Further studies on purification and identification of biogenic membrane flippase activity are under progress.
