Msp1 Extraction Assay to Study the Removal of Mislocalized TA Proteins
Published: April 30, 2023
Abstract
Source: Fresenius, H. L., Wohlever, M. L. Reconstitution of Msp1 Extraction Activity with Fully Purified Components. J. Vis. Exp. (2021).
This video demonstrates an in vitro protocol to study the Msp1-mediated removal of mislocalized TA proteins integrated into the lipid bilayer of liposomes. Msp1 and TA proteins are co-reconstituted into liposomes, and the ATP-dependent translocation of TA proteins is confirmed through affinity purification.
Protocol
1. Liposome Preparation Combine chloroform stocks of lipids in appropriate ratios to mimic the outer mitochondrial membrane. Prepare 25 mg of lipid mixture. We use a previously established mixture of lipids that mimic mitochondrial membranes, consisting of a 48:28:10:10:4 molar ratio of chicken egg phosphatidylcholine (PC), chicken egg phosphatidyl ethanolamine (PE), bovine liver phosphatidyl inositol (PI), synthetic 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and synthetic 1',3'-bi…
Disclosures
The authors have nothing to disclose.
Materials
| Biobeads | Bio-Rad | 1523920 | |
| Bovine liver phosphatidyl inositol | Avanti | 840042C | PI |
| Chicken egg phosphatidyl choline | Avanti | 840051C | PC |
| Chicken egg phosphatidyl ethanolamine | Avanti | 840021C | PE |
| Filter supports | Avanti | 610014 | |
| Glass vial | VWR | 60910L-1 | |
| Glutathione spin column | Thermo Fisher | PI16103 | |
| Mini-Extruder | Avanti | 610020 | |
| Synthetic 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine | Avanti | 840035C | DOPS |
| Synthetic 1', 3'-bis[12-dioleoyl-snglycero-3-phospho]-glycerol | Avanti | 710335C | TOCL |
| Syringe 1 mL | Norm-Ject 53548-001 | ||
| Syringe 1 mL gas-tight | Avanti 610017 |
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Cite This Article
Msp1 Extraction Assay to Study the Removal of Mislocalized TA Proteins. J. Vis. Exp. (Pending Publication), e21277, doi: (2023).