The reconstitution and fusion protocol detailed here is an efficient way to study membrane-bound proteins in a model lipid bilayer and lipid mixing by fusion proteins. Here we describe a detergent-assisted reconstitution of recombinant Drosophila atlastin, an ER fusion protein, into preformed phosphatidylcholine liposomes. Fusion of atlastin proteoliposomes is then measured by a FRET-based lipid-mixing assay.
One of the key advantages of detergent-assisted reconstitution is that the protein in detergent is not exposed to drying or solvents. We also show here that reconstitution orientation of atlastin is very efficient. Another advantage of the FRET-based lipid-mixing assay is that the liposomes do not need to be loaded with any dye, and there's no need for any extra dialyses steps.
To begin this procedure, prepare the lipid mix stocks in chloroform, as outlined in the text protocol. Add one microcurie per milliliter of DPPC to the lipid mixes, making sure to reserve at least eight microliters of this stock. Transfer the desired amount of the lipid mixes into flint glass tubes.
Then dry the lipid mixes under a gentle stream of nitrogen gas for approximately 10 minutes until no more chloroform is visible. Dry the resulting lipid film further in a desiccator by vacuuming for 30 minutes. After this, add enough A100 with 10%glycerol, two-millimolar 2-mercaptoethanol, and one-millimolar EDTA to the lipid film to bring the concentration back to 10 millimolar.
Resuspend the lipid film by vortexing lightly for 15 minutes at room temperature. Freeze the hydrated lipids in liquid nitrogen. To thaw the liposomes, let the solution sit at room temperature for 30 seconds, then transfer them back to water for faster thawing.
Repeat this freeze-thaw cycle a total of 10 times. After this, use a mini-extruder to pass the lipids through polycarbonate filters, with a pore size of 100 nanometers, 19 times. To determine the total lipid concentration of the liposomes, add four microliters of lipid stock and lysosomes to three milliliters of scintillation cocktail.
Measure the average counts per minute for stock and liposomes, and calculate the liposome concentration, as outlined in the text protocol. Store the liposomes at four degrees Celsius for up to one week. First, mix the appropriate amount of buffer, extra detergent, and protein in a 0.5-milliliter tube, as outlined in the text protocol.
Rapidly add the liposomes, and immediately vortex for five seconds to homogenize the mixture. Incubate the reaction in a nutator at four degrees Celsius for one hour. During this incubation, weigh out 0.2 grams of polystyrene adsorbent beads and transfer them to a microcentrifuge tube.
Add one milliliter of methanol to the tube, and mix for one minute to degas the beads. Then, aspirate the methanol with a 21-gauge or higher needle. To begin washing the beads, add water to them.
Let the beads mix with the water for five minutes, then aspirate the water. Repeat this water washing process four times. After this, add water to bring the polystyrene adsorbent bead slurry to a volume of one milliliter, with a final concentration of 0.2 grams per milliliter.
Calculate the amount of polystyrene adsorbent beads needed to adsorb all the detergent in each sample, as outlined in the text protocol. Cut a pipette tip to collect the bead slurry. Transfer the calculated amount of bead slurry to a 0.5-milliliter tube, and aspirate the water.
Add the samples to the tube containing the beads, and incubate in a nutator at four degrees Celsius for one hour. Transfer the samples to a fresh tube containing new beads, making sure to leave the old beads behind. Incubate again using the same conditions.
Repeat this process of transferring and incubating the beads two times. After this, transfer the sample to a new tube containing fresh beads and incubate by nutating overnight at four degrees Celsius. The next day, remove the sample from the beads and centrifuge at 16, 000 times g and four degrees Celsius for 10 minutes to pellet the insoluble protein aggregates.
Recover the supernatant, and determine the final lipid concentration by liquid scintillation counting, as outlined in the text protocol. To begin, bring the donor and acceptor proteoliposomes to a concentration of 0.15 millimolar each in A100 containing glycerol, beta-mercaptoethanol, EDTA, and magnesium chloride. Transfer each reaction to a well in a flat 96-well plate suitable for fluorescence reading, making sure to prepare at least four reactions, including a triplicate and a no-GTP negative control.
Place the plate into a preheated plate reader at 37 degrees Celsius. Measure the NBD fluorescence every minute for five minutes. Then, add five-millimolar GTP to induce fusion.
Measure the NBD fluorescence every minute for one hour using the same excitation and emission as before. After this, add five microliters of 2.5%n-Dodecyl beta-D-maltoside to the wells to dissolve the proteoliposomes, and measure the maximum NBD fluorescence every minute for 15 minutes using the same excitation and emission. In this study, recombinant Drosophila atlastin is purified and reconstituted into preformed liposomes.
The efficiency of the atlastin reconstitution is determined by floating reconstituted proteoliposomes in an iohexol discontinuous gradient. Samples of the top, middle, and bottom gradient layers are harvested and analyzed by SDS-PAGE and Coomassie staining. The quantification of the gel by densitometry shows a very high efficiency of reconstitution with negligible loses, with 96%of the total protein being found as proteoliposomes that floated to the top layer.
Less than 1%of protein is unreconstituted and found in the middle layer, and only 3%is unreconstituted or aggregated and sedimented to the bottom layer. The orientation of atlastin after reconstitution is quantified by thrombin cleavage assays. Samples are analyzed with SDS-PAGE and Coomassie staining and quantified by densitometry.
Most of the reconstituted protein is cleaved, with only 7%being protected from the protease. The kinetics and the extent of atlastin-mediated proteoliposome fusion are analyzed by lipid-mixing assays. An incubation of five minutes is performed before inducing fusion with GTP at the zero time point.
After one hour, n-Dodecyl beta-D-maltoside was added to solubilize the proteoliposomes and get the maximum fluorescent resonance transfer release. The fusion maximum in the run is seen to be 11%of maximum fluorescence. Lipids are dissolved in chloroform.
When preparing the lipid mixes, make sure to do this quickly, on a chemical hood, and on ice to avoid any evaporation. It is possible to analyze atlastin proteoliposomes by microscopy to visualize the effects of a membrane anchor on liposome shape and size. This method has been extended to study other membrane-anchored proteins and how the behave in a model lipid bilayer.
This protocol uses titrated lipids to quantify lipid mixes. Make sure to take the necessary precautions when working with radioactive materials.
