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 ...

The use of AAA-ATPases to remove proteins from a lipid bilayer is a common theme in protein quality control. A mechanistic understanding of this essential process requires a reconstituted system. Previous reconstitution with AAA-ATPases were complex and heterogeneous.Our system is simple and fully defined. This allows us to manipulate the AAA-ATPase Msp1, the substrate, and the lipid environment. To begin, add previously prepared reconstitution buffer, purified Msp1 and TA proteins and liposomes in a PCR tube, then incubate the mixture on ice for 10 minutes.During the incubation, cut the tip of a P200 pipette tip to about 1/8 of an inch in diameter. Next, vortex the tube containing BioBeads thoroughly to obtain a uniform mixture. Then, quickly remove the lid and use the cut P200 pipette tip to transfer an appropriate volume of the beads to an empty PCR tube.Once the 10-minute incubation for reconstitution is complete, using an uncut pipette tip, remove all liquid from the BioBeads. Then, transfer 100 microliters of the reconstitution into the tube with the BioBeads and allow it to rotate on a wheel for 16 hours. The following day, spin the tube in a PicoFuge to pellet the beads, then transfer the reconstituted material to a clean PCR tube and keep the tube on ice.For removing proteins that failed to reconstitute into the liposomes, equilibrate the glutathione spin columns with extraction buffer, which typically involves three rounds of washing with 400 microliters of buffer, followed by centrifugation to remove the buffer. Next, add five micromoles of each chaperone to the reconstituted material, followed by 100 microliters of extraction buffer, bringing the volume up to 200 microliters. Add this mixture to the equilibrated glutathione spin columns, then plug the spin columns and rotate them for 30 minutes, allowing the chaperones to bind to the resin.After rotation, spin the columns briefly. Collect the flow-through, which is the pre-cleared material depleted of aggregated proteins. Place it on ice and directly proceed to the extraction assay.Prepare tubes for SDS-PAGE analysis. Add 45 microliters of double distilled water to the input tube, 40 microliters of water to the flow-through tube, and 16.6 microliters of 4X SDS-PAGE loading buffer to each tube. For the extraction assay, prepare the extraction reaction and bring up the final volume to 200 microliters with the extraction buffer.Then, prewarm the extraction assay in a 30-degree Celsius heat block for two minutes. To initiate the assay, add ATP to a final concentration of two millimoles. Spin the tube for five seconds in a PicoFuge, and incubate it at 30 degrees Celsius for 30 minutes.During the incubation, take a five microliter sample of the reaction and add it to the input tube. Also, equilibrate one glutathione spin column for each sample in the extraction assay as demonstrated previously. Once the 30-minute incubation is complete, add 200 microliters of extraction buffer to the tube, bringing the total volume to 400 microliters.Then, add this reaction to the equilibrated glutathione resin and allow it to rotate for 30 minutes at four degrees Celsius. After rotation, spin the columns to collect the flow-through, then take a 10-microliter sample for the flow-through tube. Wash the resin twice with 400 microliters of extraction buffer, discarding the flow-through after each wash.After the third wash, keep the flow-through and take a 50 microliter sample for the wash tube. Next, prepare five milliliters of elution buffer by adding reduced glutathione to a final concentration of five millimoles in the extraction buffer. Add 200 microliters of the elution buffer to the spin column and incubate for five minutes.Then, centrifuge the column and collect the flow-through. Repeat the elution step once more. After the second elution, take a 50-microliter aliquot from the elution sample and add it to the elute tube.After running a western blot, the extraction efficiency can be determined by comparing the amount of substrate in the elute fraction with the input fraction. The signal in the flow-through shows some variability, but is generally similar to the input fraction, and there is no signal in the wash fraction. Typically, there is an approximately 10%extraction efficiency for the positive control and a one to 2%extraction efficiency for the negative control.When reconstitution conditions are not optimized, the extraction levels are comparable between the positive and negative samples. This technique allowed our lab to test out biophysical properties of the substrate affect recognition by Msp1.

reconstitution-msp1-extraction-activity-with-fully-purified

Research

JoVE Journal

Biochemistry

2.2K visualizações.  University of Toledo. O uso de AAA-ATPases para remover proteínas de um bicamheiro lipídico é um tema comum no controle da qualidade das proteínas. Uma compreensão mecanicista desse processo essencial requer um sistema reconstituído. A reconstituição anterior com AAA-ATPases era complexa e heterogênea.Nosso sistema é simples e totalmente definido. Isso nos permite manipular o AAA-ATPase Msp1, o substrato e o ambiente lipídulo. Para começar, adicione tampão de reconstituição previamente prepar...