The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Scaffold Liposome Preparation
NOTE: Ideally, initial experiments should use a relatively simple and featureless scaffold (comprised of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine or PC) and DGS-NTA(Ni2+) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt)). Building off of these experiments, lipid charge, flexibility, and curvature can be introduced as individual factors with the potential to alter membrane...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+into+the+understanding+of+mitochondrial+fission.">Recent advances into the understanding of mitochondrial fission.</a> Biochim Biophys Acta (BBA) - Mol Cell Res. 1833 (1), 150-161 (2013).</li><li>Los&#243;n, O. C., Song, Z., Chen, H., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fis1,+Mff,+MiD49,+and+MiD51+mediate+Drp1+recruitment+in+mitochondrial+fission.">Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission.</a> Mol Biol Cell. 24 (5), 659-667 (2013).</li><li>Otera, H., Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mff+is+an+essential+factor+for+mitochondrial+recruitment+of+Drp1+during+mitochondrial+fission+in+mammalian+cells.">Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells.</a> J Cell Biol. 191 (6), 1141-1158 (2010).</li><li>Gandre-Babbe, S., van der Bliek, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Novel+Tail-anchored+Membrane+Protein+Mff+Controls+Mitochondrial+and+Peroxisomal+Fission+in+Mammalian+Cells.">The Novel Tail-anchored Membrane Protein Mff Controls Mitochondrial and Peroxisomal Fission in Mammalian Cells.</a> Mol Biol Cell. 19 (6), 2402-2412 (2008).</li><li>Koirala, S., Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchangeable+adaptors+regulate+mitochondrial+dynamin+assembly+for+membrane+scission.">Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission.</a> Proc Natl Acad Sci. 110 (15), E1342-E1351 (2013).</li><li>Macdonald, P. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Splice+Variants+of+Dynamin-related+Protein+1+Differentially+Utilize+Mitochondrial+Fission+Factor+as+an+Effector+of+Cooperative+GTPase+Activity.">Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity.</a> J Biol Chem. 291 (1), 493-507 (2016).</li><li>Bustillo-Zabalbeitia, I., Montessuit, S., Raemy, E., Basa&#241;ez, G., Terrones, O., Martinou, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+Interaction+with+Cardiolipin+Triggers+Functional+Activation+of+Dynamin-Related+Protein+1.">Specific Interaction with Cardiolipin Triggers Functional Activation of Dynamin-Related Protein 1.</a> PLoS ONE. 9 (7), (2014).</li><li>Francy, C. 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The authors have nothing to disclose.

This protocol offers a method for investigating protein-protein interactions involving integral membrane proteins. By utilizing a modular liposome scaffold, investigators are capable of assessing the activity of one or more proteins in a lipid-proximal environment. Previous studies have demonstrated a similar method for receptor enzymes of the plasma membrane24-26. We have expanded this method to incorporate lipid cofactors and explore interactions between proteins that make up the mechan...

Studying membrane-proximal protein-protein interactions is a challenging endeavor due to difficulty in recapitulating the native environment of the integral membrane proteins involved1. This is due to the necessity of detergent solubilization and the inconsistent orientation of proteins in proteoliposomes. In order to avoid these issues, we have employed a strategy whereby soluble domains of integral membrane proteins are expressed as His-tag fusion proteins, and these soluble fragments are anchored to scaffold liposomes via interactions with NTA(Ni2+) headgroups at the lipid surface. Using these scaffolds, lipid-proximal protein interactions can be investigated over a range of lipid and protein compositions.
We have effectively applied this method to investigate the critical protein-protein interactions that govern assembly of the mitochondrial fission complex and examine lipid interactions that modulate this process2. During mitochondrial fission, a conserved membrane remodeling protein, called Dynamin-related protein 1 (Drp1)3, is recruited to the surface of the Outer Mitochondrial Membrane (OMM) in response to cellular signals that regulate energy homeostasis, apoptotic signaling, and several other integral mitochondrial processes. This large, cytosolic GTPase is recruited to the surface of mitochondria through interactions with integral OMM proteins4-8. The role of one such protein, Mitochondrial Fission Factor (Mff), has been difficult to elucidate due to an apparent weak interaction with Drp1 in vitro. Nevertheless, genetic studies have clearly demonstrated that Mff is essential for successful mitochondrial fission7,8. The method described in this manuscript was able to overcome previous shortcomings by introducing simultaneous lipid interactions that promote Drp1-Mff interactions. Overall, this novel assay revealed fundamental interactions guiding assembly of the mitochondrial fission complex and provided a new stage for ongoing structural and functional studies of this essential molecular machine.
To date, examination of interactions between Drp1 and Mff have been complicated by the inherent flexibility of Mff9, the heterogeneity of Drp1 polymers2,10, and the difficulty in purifying and reconstituting full-length Mff with an intact transmembrane domain11. We addressed these challenges by using NTA(Ni2+) scaffold liposomes to reconstitute His-tagged Mff lacking its transmembrane domain(Mff&#916;TM-His6). This strategy was advantageous because Mff&#916;TM was extremely soluble when over-expressed in E. coli, and this isolated protein was easily reconstituted on scaffold liposomes. When tethered to these lipid templates, Mff assumed an identical, outward facing orientation on the surface of the membrane. In addition to these advantages, mitochondrial lipids, such as cardiolipin, were added to stabilize Mff folding and association with the membrane11. Cardiolipin also interacts with the variable domain of Drp12,12 which may stabilize this disordered region and facilitate assembly of the fission machinery.
This robust method is widely applicable for future studies that seek to evaluate membrane-proximal protein interactions. Through the use of additional tethering/affinity interactions, the sophistication of these membrane reconstitution studies can be enhanced to mimic additional complexity found at the surface of membranes within cells. At the same time, lipid compositions can be modified to more accurately mimic the native environments of these macromolecular complexes. In summary, this method provides a means to examine the relative contributions of proteins and lipids in shaping membrane morphologies to during critical cellular processes.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylcholine (DOPC)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850375</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylethanolamine (DOPE)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:287px;">DGS-NTA(Ni2+)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">790404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Bovine Heart Cardiolipin (CL)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">840012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Chloroform</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Liposome Extruder</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">610023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cu/Rh Negative Stain Grids</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:136px;">79712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microfuge Tube</td>
			<td style="width:229px;">Beckman</td>
			<td style="width:136px;">357448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GTP</td>
			<td style="width:229px;">Jena Biosciences</td>
			<td style="width:136px;">NU-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GMP-PCP</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M3509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microtiter Plate strips</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:136px;">469949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EDTA</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">40993-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Instant Blue Coomassie Dye</td>
			<td style="width:229px;">Expedeon</td>
			<td style="width:136px;">ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HEPES</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">BP310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">BME</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">KCL</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">KOH</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Magnesium Chloride</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">223211000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">4-20% SDS-PAGE Gel</td>
			<td style="width:229px;">Bio Rad</td>
			<td style="width:136px;">456-1096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">4x Laemmli Loading Dye</td>
			<td style="width:229px;">Bio Rad</td>
			<td style="width:136px;">161-0747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HCL</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">A144S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Malachite Green Carbinol</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">229105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ammonium Molybdate Tetrahydrate</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">A7302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Laboratory Film</td>
			<td style="width:229px;">Parafilm</td>
			<td style="width:136px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Uranyl Acetate</td>
			<td style="width:229px;">Polysciences</td>
			<td style="width:136px;">21447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tecnai T12 100 keV Microscope</td>
			<td style="width:229px;">FEI</td>
			<td style="width:136px;"></td>
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			<td height="21" style="height:21px;width:287px;">Refrigerated CentriVap Concentrator</td>
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</table>

using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

While the interaction between Drp1 and Mff has been demonstrated to be important for mitochondrial fission, this interaction has been difficult to recapitulate in vitro. Our goal was to better emulate the cellular environment wherein Drp1 and Mff interact. To this end, liposomes containing limiting concentrations of NTA(Ni2+) headgroups were prepared by rehydrating a lipid film as described above. The lipid solution initially consists of unilamellar and multilamellar v...

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Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

The overall goal of this technique is to capture lipid-proximal protein-protein interactions in vitro. This method can help answer questions in the mitochondrial dynamics field, such as how protein-protein interactions at the mitochondrial outer membrane can be affected by accessory lipids. The main advantage of this technique is that tethering soluble protein domains to lipid templates ensures that a more native confirmation can be achieved.Moreover, complex interactions between soluble proteins and their tethered partners can be assessed in the presence of defined lipid cofactors. Though we use this method to investigate protein interactions in the mitochondrial fission machinery, it can also be applied to other protein complexes that localize to diverse membrane compartments. Demonstrating this procedure will be Ryan, a grad student from my laboratory.First, combine chloroform solutions of the desired lipids in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Then remove the residual solvent with the centrifugal evaporator for one hour at 37 degrees Celsius.Following this, add pre-heated buffer A to give a final lipid concentration of one to two millimolar. Incubate for 30 minutes at 37 degrees Celsius with occasional vortexing to fully resuspend the lipid mixture. After incubation, transfer the lipid mixture to a plastic test tube and place the tube in liquid nitrogen for approximately 30 seconds until completely frozen.Then place the tube in a 37 degree Celsius waterbath for one to two minutes until fully thawed. Next prepare a lipid extruder by soaking four filter supports and a polycarbonate filter in buffer A and assembling the extruder according to the manufacturer's instructions. Extrude the lipid solution through the filter 21 times using gentle constant pressure to ensure a homogenous size distribution.While extruding, use slow constant pressure to ensure liposome size homogeneity. With a one micron filter, this is less important. But with smaller filter sizes, can introduce more apparent heterogeneity.Store the extruded liposomes at four degrees Celsius. Incubate His-tagged mitochondrial fission factor or Mff with scaffold liposomes for at least 15 minutes at room temperature in buffer A and BME. For an Mff-free control, incubate the liposomes with a His-tagged control protein, such as GFP, to bind and shield exposed Nickel-NTA.Add dynamin-related protein one or Drp1 to the liposome mixture and incubate for one hour at room temperature. Next, transfer five microliters of the sample to a sheet of laboratory film and lay a carbon-coated copper-rhodium grid on top of the sample. After incubating for one minute, remove the excess liquid with filter paper and add a drop of 2%uranyl acetate.After incubating for another minute, remove the excess stain with filter paper, and transfer the sample to a grid box. On the following day, image the samples using a transmission electron microscope at the 18, 500 to 30, 000X magnification to observe ultrastructural changes in protein and liposome morphologies. Incubate His-tagged Mff and Fis1 with the scaffold liposomes for 15 minutes at room temperature in buffer A and BME.Add Drp1 and incubate each mixture for an additional 15 minutes at room temperature. Transfer the tubes to the thermocycler set to 37 degrees Celsius and initiate the reactions by adding GTP and magnesium chloride. At the desired time points, transfer 20 microliters of each reaction mixture to wells of a microtiter plate containing five microliters of 0.5 molar EDTA to chelate the magnesium and stop the reaction.Following this, prepare a set of phosphate standards by diluting potassium phosphate in buffer A and BME to calibrate the results. Add 20 microliters of each standard to the wells containing five microliters of 0.5 molar EDTA. Add 150 microliters of malachite green reagent to each well.Finally, read the OD 650 five minutes after each addition. GTP will hydrolyze slowly in the presence of the acidic malachite green reagent. So this reagent should be added to all sample wells within 10 minutes of measuring the absorbance to maximize signal to noise.In the absence in Drp1, neither Mff nor GFP resulted in membrane deformation. And only featureless liposomes were observed for GFP-decorated enriched scaffold liposomes or ESL. However, when Drp1 was added to Mff-decorated ESL templates remodeling of the liposomes was evident.Decoration of the SL with Mff enhanced GTPase activity. Conversely, when the exposed NTA head groups were blocked with His-tagged GFP, this augmented GTPase activity was ablated. Tethering of Fis1 lacking its transmembrane domain to the SL, also failed to elicit a stimulation of Drp1's GTPase activity.Similar to the SL, addition of SL/Cl to Drp1 resulted in a slight stimulation of GTPase activity that was reversed by tethering His-tagged Fis1 or GFP to the liposomes. A synergy between Mff and cardiolipin was observed as the GTPase activity of Drp1 was stimulated 2.6-fold when it was incubated with Mff-decorated SL/CL. When SL/PE/CL templates were decorated with Mff, Drp1 activity was enhanced.The ability of Drp1 to remodel liposomes into lipid tubules was enhanced by the addition of PE to the scaffold liposomes. Once mastered, these scaffolds can be prepared and used in hours if the procedure is performed properly. Following this procedure, other protein and lipid interaction methods like sedimentation and light-scattering assays can be performed in order to characterize functional interactions.The application of this technique allows people examining protein translocation to a defined biological membrane to explore transient protein interactions and the impact of specific lipid cofactors in the formation and stabilization of essential cellular assemblies.

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Biochemistry

8.7K Görüntüleme.  Case Western Reserve University School of Medicine. The overall goal of this technique is to capture lipid-proximal protein-protein interactions in vitro. This method can help answer questions in the mitochondrial dynamics field, such as how protein-protein interactions at the mitochondrial outer membrane can be affected by accessory lipids. The main advantage of this technique is that tethering soluble protein domains to lipid templates ensures that a more native confirmation can be achieved.Moreover, complex interactions between solub...