Reconstitution of Msp1 Extraction Activity with Fully Purified Components

Summary

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Abstract

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Introduction

Proper cellular function depends upon a process called proteostasis, which ensures that functional proteins are at the correct concentration and cellular location¹. Failures in proteostasis lead to compromised organelle function and are associated with many neurodegenerative diseases^2,3,4. Membrane proteins present unique challenges to the proteostasis network as they must be targeted to the correct membrane while avoiding aggregation from the hydrophobic transmembrane domains (TMDs)⁵. Consequently, specialized machinery has evo....

Protocol

1. Liposome Preparation

1. Combine chloroform stocks of lipids in appropriate ratios to mimic the outer mitochondrial membrane.
   1. 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 phosphatidyl choline (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'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (TOCL)^58,59. Sample calculations are shown in

Results

To properly interpret the results, the stain free gel and the western blot must be viewed together. The stain free gel ensures equal loading across all samples. When viewing the stain free gel, the chaperones (GST-calmodulin and GST-SGTA) will be visible in the INPUT (I) and ELUTE (E) lanes. Double check that the intensity of these bands is uniform across all of the INPUT samples. Likewise, ensure that the intensity is uniform across the ELUTE samples. The ELUTE is 5x more concentrated than the INPUT and this difference .......

Discussion

Proper mitochondrial function depends upon a robust protein quality control system. Due to inherent limits in the fidelity of the TA protein targeting pathways, mislocalized TA proteins are a constant source of stress for mitochondria. A key component of the mitochondrial proteostasis network is Msp1, which is a membrane anchored AAA+ ATPase that removes mislocalized TA proteins from the OMM. Here, we have described how to prepare proteoliposomes, co-reconstitute Msp1 and a model TA protein, and perform an extraction ass.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
ECL Select western blotting detection reagent	GE	RPN2235
Filter supports	Avanti	610014
Glass vial	VWR	60910L-1
Glutathione spin column	Thermo Fisher	PI16103
Goat anti-rabbit	Thermo Fisher	NC1050917
Mini-Extruder	Avanti	610020
Polycarbonate membrane	Avanti	610006	200 nM
PVDF membrane	Thermo Fisher	88518	45 µM
Rabbit anti-FLAG	Sigma-Aldrich	F7245
Synthetic 1,2-dioleoyl-sn-glycero-3-phospho-L-serine	Avanti	840035C	DOPS
Synthetic 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol	Avanti	710335C	TOCL
Syringe, 1 mL	Norm-Ject	53548-001
Syringe, 1 mL, gas-tight	Avanti	610017