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).

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