Name: reconstitution of msp1 extraction activity with fully purified components
Uploaded: 2021-08-10T14:00:00
Description: Watch this Scientific Journal Video about Reconstitution of Msp1 Extraction Activity with Fully Purified Components at JoVE.com

MLW developed part of this protocol during his postdoctoral studies with Dr. Robert Keenan at the University of Chicago.
This work is funded by NIH grant 1R35GM137904-01 to MLW.

1. Liposome Preparation
<ol>
	<li>Combine chloroform stocks of lipids in appropriate ratios to mimic the outer mitochondrial membrane.
	<ol>
		<li>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&#39;,3&#39;-b...

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...

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 diseases2,3,4. 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 evolved to shield the hydrophobic TMD from the cytosol and facilitate targeting and insertion into the proper cellular membrane6,7,8,9,10,11,12,13,14,15.
Mitochondria are the metabolic hub of the cell and are involved in numerous essential cellular processes such as: oxidative phosphorylation, iron-sulfur cluster generation, and apoptotic regulation16,17. These endosymbiotic organelles contain two membranes, referred to as the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM). Over 99% of the 1,500 human mitochondrial proteins are encoded in the nuclear genome and need to be translocated across one or two different membranes18,19. Proper mitochondrial function thus depends on a robust proteostasis network to correct any errors in protein targeting or translocation.
Our lab focuses on a subset of mitochondrial membrane proteins called tail-anchored (TA) proteins, which have a single transmembrane domain at the very C-terminus20,21,22,23,24. TA proteins are involved in a number of essential processes, such as apoptosis, vesicle transport, and protein translocation25. The unique topology of TA proteins requires post-translational insertion, which occurs in the endoplasmic reticulum (ER) by the Guided Entry of Tail-anchored (GET) or Endoplasmic reticulum Membrane protein Complex (EMC) pathways or into the OMM by a poorly characterized pathway20,26,27,28. The biophysical properties of the TMD are necessary and sufficient to guide TA proteins to the correct membrane29. The recognition of biophysical characteristics rather than a defined sequence motif limits the fidelity of the targeting pathways5. Thus, mislocalization of TA proteins is a common stress for the proteostasis networks. Cellular stress, such as inhibition of the GET pathway, causes an increase in protein mislocalization to the OMM and mitochondrial dysfunction unless these proteins are promptly removed30,31.
A common theme in membrane proteostasis is the use of AAA+ (ATPase Associated with cellular Activities) proteins to remove old, damaged, or mislocalized proteins from the lipid bilayer1,32,33,34,35,36,37,38. AAA+ proteins are molecular motors that form hexameric rings and undergo ATP dependent movements to remodel a substrate, often by translocation through a narrow axial pore39,40. Although great effort has been devoted to studying the extraction of membrane proteins by AAA+ ATPases, the reconstitutions are complex or involve a mixture of lipids and detergent41,42, which limits the experimental power to examine the mechanism of substrate extraction from the lipid bilayer.
Msp1 is a highly conserved AAA+ ATPase anchored in the OMM and peroxisomes that plays a critical role in membrane proteostasis by removing mislocalized TA proteins43,44,45,46,47. Msp1 was also recently shown to alleviate mitochondrial protein import stress by removing membrane proteins that stall during translocation across the OMM48. Loss of Msp1 or the human homolog ATAD1 results in mitochondrial fragmentation, failures in oxidative phosphorylation, seizures, increased injury following stroke, and early death31,49,50,51,52,53,54,55,56.
We have shown that it is possible to co-reconstitute TA proteins with Msp1 and detect the extraction from the lipid bilayer57. This simplified system uses fully purified proteins reconstituted into defined liposomes which mimic the OMM (Figure 1)58,59. This level of experimental control can address detailed mechanistic questions of substrate extraction that are experimentally intractable with more complex reconstitutions involving other AAA+ proteins. Here, we provide experimental protocols detailing our methods for liposome preparation, membrane protein reconstitution, and the extraction assay. It is our hope that these experimental details will facilitate further study of the essential but poorly understood process of membrane proteostasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biobeads</td>
			<td>Bio-Rad</td>
			<td>1523920</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine liver phosphatidyl inositol</td>
			<td>Avanti</td>
			<td>840042C</td>
			<td>PI</td>
		</tr>
		<tr>
			<td>Chicken egg phosphatidyl choline</td>
			<td>Avanti</td>
			<td>840051C</td>
			<td>PC</td>
		</tr>
		<tr>
			<td>Chicken egg phosphatidyl ethanolamine</td>
			<td>Avanti</td>
			<td>840021C</td>
			<td>PE</td>
		</tr>
		<tr>
			<td>ECL Select western blotting detection reagent</td>
			<td>GE</td>
			<td>RPN2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter supports</td>
			<td>Avanti</td>
			<td>610014</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>VWR</td>
			<td>60910L-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione spin column</td>
			<td>Thermo Fisher</td>
			<td>PI16103</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>NC1050917</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Extruder</td>
			<td>Avanti</td>
			<td>610020</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>Avanti</td>
			<td>610006</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Thermo Fisher</td>
			<td>88518</td>
			<td>45 &#181;M</td>
		</tr>
		<tr>
			<td>Rabbit anti-FLAG</td>
			<td>Sigma-Aldrich</td>
			<td>F7245</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic 1,2-dioleoyl-sn-glycero-3-phospho-L-serine</td>
			<td>Avanti</td>
			<td>840035C</td>
			<td>DOPS</td>
		</tr>
		<tr>
			<td>Synthetic 1&#39;,3&#39;-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol</td>
			<td>Avanti</td>
			<td>710335C</td>
			<td>TOCL</td>
		</tr>
		<tr>
			<td>Syringe, 1 mL</td>
			<td>Norm-Ject</td>
			<td>53548-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 mL, gas-tight</td>
			<td>Avanti</td>
			<td>610017</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of msp1 extraction activity with fully purified components

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.

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 ...

Watch this Scientific Journal Video about Reconstitution of Msp1 Extraction Activity with Fully Purified Components at JoVE.com

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

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

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

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