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.

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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Biochemistry

2.3K Views.  University of Toledo. Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Video: Reconstitution of Msp1 Extraction Activity with Fully Purified Components

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.