One of the key advantages of this technique for characterizing membrane transporters is that you can determine the relative affinity of the transporter for specific substrates, as well as gain insight on the mechanism of transport. The characterization of membrane proteins that use protein gradients to drive substrate transport is particularly suited for this inside-out vesicle assay that we demonstrate here. While this assay requires some specialized equipment such as a French press and access to an ultracentrifuge, we think it may be technically easier than assays that require membrane transporters to be reconstituted into artificial liposomes.
The main advantage of this technique is that it enables us to quantify the relative affinities for different substrates and do competition assays to test the effect of competing substrates on transport grates. In the case of the BAT1 transporter used to illustrate this assay, we were also able to show substrate activation of the transporter by arginine. To begin this procedure, culture the prepared E.coli mutant cells that express the target protein by inoculating a single colony in five milliliters of polyamine 3 media with appropriate antibiotics and growing it overnight at 37 degrees Celsius.
The next day, transfer this culture to 500 milliliters of fresh polyamine 3 media that is supplemented with 0.0002%arabinose and grow until the cell culture reaches an OD600 of between 0.6 and 0.8. After this, centrifuge at 2000 times G and at four degrees Celsius for 15 minutes to collect the cell pellet. Re-suspend the pellet and wash it three times with buffer one by centrifuging at 2000 times G and at four degrees Celsius for 15 minutes each time.
The final volume of cells in Buffer one after the third spin should be 10 milliliters. Set out a 35 milliliters French pressure cell and pour the cell suspension into the body of the cell. Turn the machine on with the switch on the RHS at the back of the unit and set the ratio selector switch to high.
Turn the pump switch on. The piston will begin to rise from the base to displace the air in the chamber. Increase the pressure by turning the pressure increase valve clockwise until the gauge reaches 640 PSI.
This will create an internal pressure of 10, 000 PSI. Once the cell has reached target pressure, open the flow valve assembly slightly by turning it counterclockwise. Adjust the opening of the valve to allow a flow rate of only about 10 drops per minute.
When the stop line on the piston body reaches the top of the flow cell switch the pump switch off. Lower the bottom plate by placing the ratio selector switch to down, and then, setting the pump switch on. When the bottom plate is fully retracted, reduce the system pressure to zero by turning the pressure increase valve fully counterclockwise.
Turn the pump switch off and turn the machine off. Centrifuge the French press eluant at 10, 000 times G and at four degrees Celsius for 15 minutes to remove unbroken cells and cell debris. Discard the pellet and transfer the supernatant to ultracentrifuge tubes.
Ultracentrifuge the resulting supernatant at 150, 000 times G and at four degrees Celsius for one hour to pellet the membrane vesicles. Wash the membrane vesicles once without re-suspension in buffer two. Then, use a Dounce tissue grinder to re-suspend the membrane vesicles in buffer two.
Prepare 100 microliter aliquots of the membrane preparations in 1.5 milliliter centrifuge tubes and store them at minus-80 Celsius. First, wash and re-suspend the vesicles in 30 millimolar of Tris at pH 7.8, that contains 0.1 millimolar of cobalt II chloride. Add one microliter of carboxypeptidase A in sodium chloride, Tris, and cobalt II chloride to each 100 microliter sample.
Incubate at 20 degrees Celsius for 20 minutes. Add five microliters of a solution containing sodium EDTA and 2-Mercaptoethanol at PH 7.5 to stop the digestion. Incubate the solution at room temperature for one hour to fully inactivate the enzyme.
Next, add between five and 10 microliters of a solution containing lithium dodecyl sulfate, glycerol, bromophenol blue, and Tris at pH 7.5 to 100 microliters of the sample and analyze samples by electrophoresis. To perform amino blotting, lay a nitrocellulose filter moistened with 25 millimolar sodium hydrogen phosphate upon the polyacrylamide gel. Place these between two moistened cellulose filters, and finally, between two moistened plastic scouring pads.
Place this assemblage between the electrodes of a chamber containing 25 millimolar sodium hydrogen phosphate at a temperature between two and four degrees Celsius. Electrotransfer the proteins at 20 volts and at two to three amps for three hours. Block the nitrocellulose membrane in blocking buffer overnight at two degrees Celsius.
The next day, incubate the nitrocellulose at 20 milliliters of one to 5, 000 dilution of Anti-His C-terminal HRP antibody and blocking buffer for two hours and wash twice with 50 milliliters of buffer for a total of 60 minutes. To visualize the amino blot, dissolve six milligrams of 4-Chloro-1-naphthol in 20 milliliters of denatured alcohol and add 80 milliliters of 15 millimolar Tris and 50 microliters of 30%hydrogen peroxide. Bathe the filter paper in this substrate solution for 20 minutes.
When sufficient color has developed, rinse the membrane with water and allow it to dry. To begin, incubate the 100 microliter aliquot of membrane vesicles at 12 degrees Celsius for five minutes. Add buffer 3 containing radiolabeled polyamines at a final concentration of 50 micromolar to the membrane vesicles in 1.5 milliliters microcentrifuge tubes to initiate transport.
Conduct the transport assay at 12 degrees Celsius for one minute. After this, transfer the reaction mixtures to the filtration manifold and filter it through a 0.45 micrometer nitrocellulose membrane filter. Add three milliliters of ice-cold assay buffer containing a tenfold higher concentration of unlabeled polyamines, followed by three milliliters of assay buffer without the polyamines to reduce non-specific binding.
Transfer the washed filters to 20 milliliter disposable scintillation vials containing 10 milliliters of scintillation liquid, and use a liquid scintillation counter to determine the radioactivity. Calculate the net polyamine uptake as outlined in the text protocol. Determine the Km for the substrate by measuring the uptake of radiolabeled substrate into vesicles expressing the target protein and calculate the Michaelis-Menten kinetics using a nonlinear regression method.
For the competition experiments, add the non-labeled competitive substrate made in the assay buffer to the centrifuge tube containing 100 microliters of vesicles at 12 degrees Celsius while simultaneously adding 50 micromolar of the radiolabeled polyamine. Measure the radioactivity trapped inside vesicles as previously described. Determine a parent KM for the competitive substrates by measuring the uptake of radiolabeled polyamines and the presence of 100 micromolar or higher non-labeled substrate and use a nonlinear regression method to plot the curve.
In this study, an anti-porter is characterized by first expressing the protein in E.coli, and then, generating membrane vesicles so that the heterologously-expressed protein can be assayed in a cell-free system. A western blot is used to verify that AtBAT1 is translocated to vesicle. Probing the blot with an Anti-His C-terminal antibody revealed a fusion construct protein that is approximately 72.3 kilodaltons.
Digestion of the vesicles prior to SDS-PAGE resulted in a diminution, but not a complete loss of the probe signal. At 12 degrees Celsius, uptake a radiolabeled spermidine by the vesicles is highest at one minute and remained linear over three minutes. Therefore, the incubation time for the transport assay is fixed at one minute.
After one minute, there is no net uptake of isotope by membrane vesicles that were prepared and stored at pH 8.0 as there is no proton gradient across the vesicle membrane. To demonstrate the effect of dissipation of the artificial proton gradient, the membrane vesicles are incubated in pH 8.0 buffer for 10 minutes prior to the addition of labeled substrate, leading to a minimal uptake of radiolabeled substrate. Taken together, these results indicate that the proton-driven uptake of spermidine is due to the BAT1 protein.
To determine the substrate specificity of the protein, Km values are calculated by measuring the uptake of radiolabeled substrate at different concentrations. The Km values for spermidine, putrescine, and arginine indicate that this protein is a high-affinity polyamine and arginine exchanger. Affinity of the transporter for a particular substrate can also be determined indirectly by using competition assays.
These assays reveal that GABA is a competitive inhibitor of spermidine. Furthermore, measuring the uptake of 50 micromolar of radiolabeled spermidine in the presence of varying concentrations of different amino acids reveals that AtBAT1 is also capable of transporting glutamate and alanine at millimolar concentrations. When performing this procedure, the integration of the membrane transporter into the bacterial membrane is, obviously, critical.
Verification that the protein of interest is integrated into the membrane can be done using antibodies directed against the C-terminal tags. Genetic variation of human transporters can affect the transport of, both, metabolites and drugs that are substrates of the particular transporter. Thus, allelic variation can affect, both, uptake and excretion of drugs.
Many allelic variants can be quickly made by side-directed mutagenesis of the transporter gene in an expression vector. Functional assays of these variants can then be tested under very controlled conditions using inside-out vesicle assays. Membrane transporters constitute eight to 10 of all proteins and the majority have not been characterized completely in any of the model organisms.
Transporters that are localized to organelle membranes are particularly challenging to characterize by heterologous expression in eukaryotic cells. If exchange type or proton-mediated transporters can be localized to this E.coli membrane, these transporters can be functionally-characterized using this in vitro assay. The quality and yield of vesicles is affected by the rate of elution of cell fragments from the French press.
In doing transport assays of vesicles with isotopes, it is important to have an experimental setup that facilitates doing experimental replicates. There are many ways of doing this, but we think it will be helpful for us to show you how we have done it.
