Msp1 Extraction Assay to Study the Removal of Mislocalized TA Proteins

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.