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 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.
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.
1. Liposome Preparation
2 Reconstitution of Msp1 and Model TA protein
3. 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 in intensity will be visible in the gels.
After using the stain free gel to confirm proper loading, examine the western blot to determine extraction activity. Measure extraction activity by comparing the amount of substrate in the ELUTE (E) fraction relative to the INPUT (I) fraction. The signal in the Flow Through (FT) shows some variability, but is generally similar to the INPUT fraction. There should be no signal in the WASH (W) fraction. Typically, there is ~10% extraction efficiency for the positive control and 1-2% extraction efficiency in the negative control (Figure 2). Recall that the ELUTE fraction is 5x as intense as the INPUT fraction, so this needs to be taken into account when judging extraction efficiency. If reconstitution conditions are not optimized, there is typically comparable extraction levels in both the + ATP and – ATP samples (Figure 3). This result is attributed to a failure of Msp1 to efficiently reconstitute, resulting in numerous proteoliposomes without a functional Msp1 hexamer.
Lipid | Mole % | MW | Avg. MW | µmol in 25 mg | mg in 25 mg | Chlor stock (mg/mL) | µL for 25 mg |
PC | 48% | 770 | 369.6 | 14.82 | 11.41 | 25 | 456.4 |
PE | 28% | 746 | 208.88 | 8.64 | 6.45 | 25 | 258.0 |
PI | 10% | 902 | 90.2 | 3.09 | 2.78 | 10 | 278.5 |
DOPS | 10% | 810 | 81 | 3.09 | 2.50 | 10 | 250.1 |
TOCL | 4% | 1502 | 60.08 | 1.23 | 1.85 | 25 | 74.2 |
Conc. Corr Avg MW | 809.76 | ||||||
µmol in 25 mg | 30.87 |
Table 1: Sample calculations for liposome preparation. The key goals of this table are to calculate the concentration corrected average molecular weight of the lipid mixture and the volume of each lipid stock required to make the liposomes. MW and stock concentration come from product labels. Lipids and mole % are chosen by user.
Figure 1: Cartoon of extraction assay and list of key steps. Please click here to view a larger version of this figure.
Figure 2: Representative data showing a properly functioning assay. Extraction efficiency is determined by comparing the amount of substrate in the ELUTE fraction with the INPUT fraction. Recall that the gel has 5x higher loading of the ELUTE fraction relative to the INPUT fraction. Please click here to view a larger version of this figure.
Figure 3: Representative data of a failed reconstitution and extraction assay. Here, the activity in the + ATP sample is comparable to the activity in the – ATP sample. Please click here to view a larger version of this figure.
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 assay. We previously used this assay to demonstrate that Msp1 directly recognizes mislocalized TA proteins and is capable of extracting these proteins from a lipid bilayer without any accessory proteins or cofactors57.
A drawback of the assay is that there is some variability in the reconstitution process. To control for prep-to-prep variability, we always include a positive and negative control on the same gel/western blot to ensure that our assay is working as intended. We avoid making comparisons between reconstitutions that were done on different days or drawing comparisons between different western blots. The only comparisons we make are for samples reconstituted in parallel and run on the same gel/western blot. We are also quite conservative in our data interpretation. Although it is possible to quantify extraction efficiency using ImageJ, we typically describe our experiments as having full activity, intermediate activity, or no activity.
One source of variability is the total amount of protein reconstituted. While the majority of unincorporated proteins are removed by the pre-clearing process, the less than perfect reconstitution efficiency of Msp1 can have an outsized effect on the observed efficiency of substrate extraction. This effect arises from the fact that Msp1 functions as a homohexamer, but purifies as a monomer57. Liposomes containing anything other than 6x copies of Msp1 will be inactive. For example, a liposome with only 5 copies of Msp1 will be inactive. The only way to form stable full-length Msp1 hexamers is to inactivate ATPase activity with non-hydrolyzable ATP analogs (ATPγS) or the inactivating E193Q Walker B mutant, neither of which are compatible with an activity assay. Overcoming this technical hurdle is an area of active research in our lab.
Another area of active research focuses on making the extraction assay more quantitative. The current method relies on pull downs and western blotting for signal detection, both of which are only semi-quantitative and show assay to assay variability. Covalent modification of extracted substrates would eliminate the variability that arises from the pull downs. Likewise, use of radioactive or fluorescent labels on substrates would eliminate the need for western blots and the associated variability.
A major strength of the assay is that the system is completely defined. The membrane proteins are recombinantly expressed and purified and it is possible to make defined mutants in both Msp1 and the substrate to study specific aspects of the reaction. The role of the lipid environment in proteostasis has been largely ignored due to the technical challenges of studying this in a detailed manner. Because our assay uses liposomes with a defined lipid composition, this allows for full experimental control of the lipid environment. We can easily modulate factors such as: lipid fluidity, bilayer thickness, headgroup identity, and liposome size. We are actively working to use our assay to examine the role of the lipid environment on Msp1 activity. It is our hope that the in vitro reconstitution and extraction assay described here can serve as a simplified model system to study the common cellular process of AAA+ ATPase mediated extraction of membrane proteins from a lipid bilayer.
The authors have nothing to disclose.
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.
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 |