The use of AAA-ATPases to remove proteins from a lipid bilayer is a common theme in protein quality control. A mechanistic understanding of this essential process requires a reconstituted system. Previous reconstitution with AAA-ATPases were complex and heterogeneous.
Our system is simple and fully defined. This allows us to manipulate the AAA-ATPase Msp1, the substrate, and the lipid environment. To begin, add previously prepared reconstitution buffer, purified Msp1 and TA proteins and liposomes in a PCR tube, then incubate the mixture on ice for 10 minutes.
During the incubation, cut the tip of a P200 pipette tip to about 1/8 of an inch in diameter. Next, vortex the tube containing BioBeads thoroughly to obtain a uniform mixture. Then, quickly remove the lid and use the cut P200 pipette tip to transfer an appropriate volume of the beads to an empty PCR tube.
Once the 10-minute incubation for reconstitution is complete, using an uncut pipette tip, remove all liquid from the BioBeads. Then, transfer 100 microliters of the reconstitution into the tube with the BioBeads and allow it to rotate on a wheel for 16 hours. The following day, spin the tube in a PicoFuge to pellet the beads, then transfer the reconstituted material to a clean PCR tube and keep the tube on ice.
For removing proteins that failed to reconstitute into the liposomes, equilibrate the glutathione spin columns with extraction buffer, which typically involves three rounds of washing with 400 microliters of buffer, followed by centrifugation to remove the buffer. Next, add five micromoles of each chaperone to the reconstituted material, followed by 100 microliters of extraction buffer, bringing the volume up to 200 microliters. Add this mixture to the equilibrated glutathione spin columns, then plug the spin columns and rotate them for 30 minutes, allowing the chaperones to bind to the resin.
After rotation, spin the columns briefly. Collect the flow-through, which is the pre-cleared material depleted of aggregated proteins. Place it on ice and directly proceed to the extraction assay.
Prepare tubes for SDS-PAGE analysis. Add 45 microliters of double distilled water to the input tube, 40 microliters of water to the flow-through tube, and 16.6 microliters of 4X SDS-PAGE loading buffer to each tube. For the extraction assay, prepare the extraction reaction and bring up the final volume to 200 microliters with the extraction buffer.
Then, prewarm the extraction assay in a 30-degree Celsius heat block for two minutes. To initiate the assay, add ATP to a final concentration of two millimoles. Spin the tube for five seconds in a PicoFuge, and incubate it at 30 degrees Celsius for 30 minutes.
During the incubation, take a five microliter sample of the reaction and add it to the input tube. Also, equilibrate one glutathione spin column for each sample in the extraction assay as demonstrated previously. Once the 30-minute incubation is complete, add 200 microliters of extraction buffer to the tube, bringing the total volume to 400 microliters.
Then, add this reaction to the equilibrated glutathione resin and allow it to rotate for 30 minutes at four degrees Celsius. After rotation, spin the columns to collect the flow-through, then take a 10-microliter sample for the flow-through tube. Wash the resin twice with 400 microliters of extraction buffer, discarding the flow-through after each wash.
After the third wash, keep the flow-through and take a 50 microliter sample for the wash tube. Next, prepare five milliliters of elution buffer by adding reduced glutathione to a final concentration of five millimoles in the extraction buffer. Add 200 microliters of the elution buffer to the spin column and incubate for five minutes.
Then, centrifuge the column and collect the flow-through. Repeat the elution step once more. After the second elution, take a 50-microliter aliquot from the elution sample and add it to the elute tube.
After running a western blot, the extraction efficiency can be determined by comparing the amount of substrate in the elute fraction with the input fraction. The signal in the flow-through shows some variability, but is generally similar to the input fraction, and there is no signal in the wash fraction. Typically, there is an approximately 10%extraction efficiency for the positive control and a one to 2%extraction efficiency for the negative control.
When reconstitution conditions are not optimized, the extraction levels are comparable between the positive and negative samples. This technique allowed our lab to test out biophysical properties of the substrate affect recognition by Msp1.
