Msp1 Extraction Assay to Study the Removal of Mislocalized TA Proteins

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…

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