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 ...

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

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 ...

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 ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

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.

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...