This study investigates the role of Msp1, an ATPase in the mitochondrial outer membrane, in the removal of mislocalized tail-anchored proteins. The research focuses on the mechanism of translocation of these proteins through Msp1's central pore, utilizing liposome reconstitution and affinity purification techniques.
Msp1, an ATPase in the mitochondrial outer membrane, removes mislocalized tail-anchored, or TA, proteins from the membrane via its hexameric central pore, facilitating their degradation.
To understand the mechanism of translocation, co-reconstitute TA proteins, and Msp1 into liposomes, enabling their integration into the lipid bilayer.
Prepare a reaction mix by adding the reconstituted liposomes to a suitable buffer containing chaperones — cytosolic quality control proteins. The chaperones are tagged with the glutathione-S-transferase or GST enzyme.
Add adenosine triphosphate — ATP — to the reaction. The Msp1 binds to the ATP molecules and hydrolyzes them. Using hydrolysis-generated energy, Msp1 translocates the membrane-bound TA proteins through its central pore. The chaperones in the reaction mix bind to the transmembrane domain of the extracted TA proteins.
Next, load the reaction mix onto an affinity purification column. The column contains a resin conjugated with reduced glutathione or GSH. The GST protein on the chaperones binds to the conjugated GSH — its substrate — attaching the chaperone-TA protein complexes to the resin.
Spin the column to remove the TA proteins still integrated into liposomes. Now, add an elution buffer containing an excess of GSH. The free GSH molecules competitively displace the binding between GST and conjugated GSH.
Elute the released chaperone-TA protein complexes with the buffer. Analyze the eluate to confirm the presence of TA proteins, denoting their removal from the liposomes.
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, pre-warm the extraction assay in a 30-degree degree Celsius heat block for two minutes.
To initiate the assay, add ATP to a final concentration of 2 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 5-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.
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 milliliters 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 5 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.
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