Name: An In Vitro Assay For LpxC Degradation By The Membrane-Bound Protease FtsH And Adaptor LapB From Escherichia coli (Video) | JoVE Journal
Uploaded: 2026-06-22T13:00:00.000+00:00
Duration: 11 min 5 s
Description: Learn a fluorescence-based in vitro assay for LpxC degradation by the membrane-bound protease FtsH and adaptor LapB from Escherichia coli. This sensitive method enables quantitative kinetic analysis of membrane-associated proteolysis.

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All research was conducted at Yale University. W.M. is supported by the National Institute of General Medical Sciences under award numbers R01GM137068 and RM1GM149406, and by the Richard and Susan Smith Family Foundation through the Odyssey Award.

1. Labeling LpxC with Atto488
<ol>
	<li>LpxC preparation
	<ol>
		<li>Express and purify LpxC protein as reported previously18. Exchange the buffer of purified LpxC from Tris to 50 mM phosphate buffer, pH 7.6 ( 6 mM NaH2PO4, 44 mM Na2HPO4, 150 mM NaCl) by gel filtration using a high-resolution gel filtration column (e.g., Superdex 200 Increase 10/300 GL column).</li>
		<li>Concentrate LpxC using an ultrafiltration column to a final concentration of at least 5 mg/mL at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Labeling LpxC with Atto488
	<ol>
		<li>Dilute 0.5 mg of LpxC in 50 mM phosphate buffer, pH 7.6, to a final volume of 225 &#181;L. Add 25 &#181;L of 1 M sodium bicarbonate to adjust the pH to approximately 8.3.</li>
		<li>Dissolve 0.24 mg of Atto488 reactive dye in 20 &#181;L of 0.1 M sodium bicarbonate buffer to prepare a stock solution (0.012 mg/&#181;L). Prepare a working solution by diluting 2 &#181;L of the stock solution with 18 &#181;L of 0.1 M sodium bicarbonate buffer, yielding a final concentration of 0.0012 mg/&#181;L.</li>
		<li>Immediately add 4 &#181;L of the Atto488 working solution to the protein solution (corresponding to a molar ratio of LpxC: Atto488 of 3:1). Wrap the tube with aluminum foil and incubate at 4 &#176;C for 2 h with gentle agitation (18 rpm). 
		NOTE: Both the Atto488 stock and working solutions can be flash-frozen in liquid nitrogen and stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Separation of LpxC&#8211;Atto488 from free dye
	<ol>
		<li>Equilibrate a PD-10 desalting column with 10 mL of ultrapure water, followed by 15 mL of 50 mM phosphate buffer, pH 7.6.</li>
		<li>Apply the labeling reaction mixture to the column and allow it to fully enter the resin.</li>
		<li>Elute with 5 mL of 50 mM phosphate buffer, pH 7.6. Two distinct bands corresponding to LpxC&#8211;Atto488 (first band) and free Atto488 dye (second band) should become visible. Collect fractions from the first band in ~0.5 mL aliquots.</li>
		<li>Analyze eluted fractions by SDS-PAGE. Visualize fluorescence under UV illumination before Coomassie Brilliant Blue (CBB) staining.</li>
		<li>Pool fractions containing LpxC&#8211;Atto488 and concentrate the labeled protein to approximately 2 mg/mL.</li>
		<li>Estimate labeling efficiency by measuring the protein concentration and Atto488 concentration using a microvolume spectrophotometer. 
		NOTE: A ratio of Atto488 concentration (in pmol/&#181;L) to LpxC concentration (in mg/mL) in the range of 2&#8211;5 indicates an appropriate labeling level. This corresponds to a molar ratio of Atto488 to LpxC ranging from 1:15 to 1:6 after labeling.</li>
		<li>Aliquot LpxC&#8211;Atto488 into 1.5 mL microcentrifuge tubes, flash-freeze in liquid nitrogen, and store at -80 &#176;C. &#65279;At this point, the protocol can be paused for up to 3 months. 
		CAUTION: Liquid nitrogen is extremely cold; handle it with proper protective equipment (cryo-gloves, eye protection).</li>
	</ol>
	</li>
</ol>
2. Reconstitution of FtsH/LapB into proteoliposomes
<ol>
	<li>Liposomes preparation
	<ol>
		<li>Transfer 100 mg of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in chloroform into a glass test tube. Evaporate the solvent under a gentle stream of N&#8322;, followed by overnight drying under vacuum. 
		NOTE: POPC has a phase transition temperature of ~-2 &#176;C and is widely used in membrane biophysics, liposome formulations, and cell model systems. 
		CAUTION: Chloroform can harm the eyes, skin, liver, kidneys, and nervous system. Proper protective equipment (gloves, eye protection, mask) is required during handling. Handle it in a hood using a glass syringe.</li>
		<li>Add 1,315 &#181;L of buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl) to the dried POPC film. Resuspend the lipids by alternating 1 min of water-bath sonication with 1 min of vortexing at maximum speed for a total of ~15 min, or until fully dispersed. (final lipid concentration: 100 mM). 
		NOTE: To improve lipid resuspension, adjust the immersion depth of the glass test tube in the water-bath sonicator to maximize agitation. Resuspended lipids can be stored at -20 &#176;C. At this point, the protocol can be paused for up to 1 month.</li>
		<li>Aliquot the liposome suspension into 200 &#181;L portions and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Liposomes extrusion
	<ol>
		<li>Thaw a 200 &#181;L liposome aliquot in a water bath at room temperature and dilute with 800 &#181;L of buffer A to a final volume of 1 mL.</li>
		<li>Subject the liposomes to three freeze-thaw cycles by alternating between liquid nitrogen and a water bath at room temperature.</li>
		<li>Rinse syringes, two filter supports, and a 200 nm polycarbonate membrane with buffer A. Assemble the mini-extruder and extrude the liposomes 20 times. 
		NOTE: The liposome suspension should appear transparent after extrusion.</li>
		<li>Divide the 1 mL extruded liposome suspension into three tubes (330 &#181;L per tube) and dilute each with 600 &#181;L of buffer A.</li>
	</ol>
	</li>
	<li>Triton X-100 swelling titration 
	NOTE: Triton X-100 is a &#65279;low critical micelle concentration (CMC) detergent, and &#65279;it has proven to be most successful in many cases21 
	CAUTION: Triton X-100 is a hazardous chemical, primarily causing severe eye damage, skin irritation, and harmful effects if swallowed. Proper protective equipment (gloves, eye protection) is required during handling.
	<ol>
		<li>Prepare 10% (v/v) Triton X-100 by mixing 100 &#181;L Triton X-100 with 900 &#181;L ultrapure water, and incubating it in a shaker to fully dissolve Triton X-100.</li>
		<li>Measure the absorbance of liposomes at 540 nm (A&#8325;&#8324;&#8320;) using a spectrophotometer.</li>
		<li>Add 5 &#181;L aliquots of 10% Triton X-100 sequentially, gently mixing after each addition, and measure A&#8325;&#8324;&#8320;. Continue adding Triton X-100 until A&#8325;&#8324;&#8320; reaches a maximum, then add Triton X-100 an additional 4&#8211;5 times until the absorbance decreases to approximately 60% of the peak value.</li>
		<li>Based on the titration curve, swell liposomes by adding 35 &#181;L of 10% Triton X-100 and incubate at room temperature for 1 h with gentle agitation.</li>
	</ol>
	</li>
	<li>Reconstitution of FtsH/LapB into liposomes
	<ol>
		<li>Express and purify FtsH and LapB proteins as described previously18.</li>
		<li>Wash ~2 g of a nonpolar polystyrene adsorbent (e.g., Bio-Beads SM-2) once with 10 volumes of methanol, once with 10 volumes of ethanol, and ten times with 10 volumes of ultrapure water. Store the washed adsorbent in buffer A at 4 &#176;C.</li>
		<li>For each tube of Triton X-100&#8211;swollen liposomes, add 3 &#181;L of 500 mM tris(2-carboxyethyl)phosphine (TCEP), 22.5 &#181;g of FtsH, and 22.5 &#181;g of LapB. Adjust the total volume to 1 mL with buffer A and incubate at RT for 1 h with gentle agitation. 
		NOTE: &#65279;Protein-to-lipid ratios of 1:20 to 1:200 (wt/wt) were &#65279;routinely used during proteoliposomes reconstitution21. Here, a ratio of 1:100 is used.</li>
		<li>Remove detergent by sequential incubation with nonpolar polystyrene adsorbent: 40 mg at RT for 0.5 h, followed by 40 mg at RT for 1 h, then 40 mg at 4 &#176;C overnight, and finally 40 mg at 4 &#176;C for 1 h.</li>
		<li>Briefly centrifuge to pellet the nonpolar polystyrene adsorbent at 4 &#176;C (400 &#215; g for 3 s). Transfer the supernatant to ultracentrifuge tubes. Wash the nonpolar polystyrene adsorbent with 1 mL of buffer A and combine the wash with the supernatant. Add additional buffer A to a final volume of ~6 mL. 
		NOTE: For safety, ultracentrifuge tubes must contain at least 6 mL of liquid.</li>
		<li>Balance the tubes and ultracentrifuge at 250,000 &#215; g at 4 &#176;C for 45 min in a fixed-angle rotor (e.g., MLA-80) to pellet proteoliposomes.</li>
		<li>Discard the supernatant and resuspend the proteoliposome pellet in 70 &#181;L of buffer A supplemented with 1 mM&#160;TCEP. 
		NOTE: The final proteoliposome volume is approximately 100 &#181;L.</li>
		<li>Flash-freeze the proteoliposomes in liquid nitrogen and store at -80 &#176;C. &#65279; 
		NOTE: At this point, the protocol can be paused for up to 1 month.</li>
	</ol>
	</li>
	<li>SDS-PAGE analysis of proteoliposomes
	<ol>
		<li>Mix proteoliposomes thoroughly and combine 5 &#181;L of the sample with 5 &#181;L of 2&#215; SDS loading buffer (0.1 M Tris-HCl, pH 6.8, 5% SDS, 20% glycerol, 0.01% bromophenol blue, 200 mM DTT).</li>
		<li>Load proteoliposome samples alongside 525 ng of FtsH (corresponding to 0.06 &#181;M FtsH hexamer in 20 &#181;L) on a 4&#8211;20% SDS-PAGE gel.</li>
		<li>Stain the gel with CBB.</li>
		<li>Scan the gel and quantify the band intensity of FtsH for each sample. Calculate the volume of proteoliposome suspension equivalent to 525 ng of FtsH.</li>
	</ol>
	</li>
</ol>
3. In vitro degradation of LpxC&#8211;Atto488
<ol>
	<li>Perform in vitro degradation assays in a 20 &#181;L reaction containing FtsH/LapB proteoliposomes (corresponding to 0.06 &#181;M FtsH) and 0.5, 1, 2, 2.5, 5, 10, 20, 40 &#181;M LpxC-Atto488 in degradation buffer (50 mM Tris-acetate, pH 8.0, 10 mM Mg-acetate, 5 mM ATP, 25 &#181;M Zn-acetate, 80 mM NaCl, 1.4 mM &#946;-mercaptoethanol).</li>
	<li>For the measurement of each concentration of substrate, prepare a reaction of 70 &#181;L.
	<ol>
		<li>Take a 20 &#181;L fraction and quench it by immediately mixing it with 48 &#181;L of 5% trichloroacetic acid (TCA) in 1.5 mL microcentrifuge tubes (0 min). Take the second and the third fractions and quench them at 10 and 20 min, respectively.</li>
		<li>In parallel, prepare corresponding control reactions lacking proteoliposomes for background subtraction. 
		NOTE: Three replicate measurements are recommended in order to obtain reliable results. 
		Minimize light exposure when handling LpxC&#8211;Atto488. 
		CAUTION: TCA is a highly corrosive chemical that causes severe skin burns, permanent eye damage, and intense respiratory irritation upon inhalation. Handle it with proper protective equipment (gloves, eye protection, mask).</li>
	</ol>
	</li>
	<li>Incubate samples at 37 &#176;C for 30 min to precipitate proteins, then centrifuge at 18,800 &#215; g at room temperature for 20 min.</li>
	<li>Neutralize samples by transferring 50 &#181;L of supernatant into a tube containing 60 &#181;L of 500 mM Tris-HCl,&#160;pH 8.8. Mix well and transfer 100 &#181;L to a black 96-well plate. Measure fluorescence using a plate reader with excitation at 498 nm and emission at 520 nm.</li>
</ol>

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<li>Chai-Danino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane-embedded+polar+residues+target+membrane+proteins+for+degradation+by+the+quality+control+protease+FtsH%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Membrane-embedded polar residues target membrane proteins for degradation by the quality control protease FtsH.</a> bioRxiv. , (2023).</li>
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<li>Westphal, K., Langklotz, S., Thomanek, N., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+trapping+approach+reveals+novel+substrates+and+physiological+functions+of+the+essential+protease+ftsh+in+Escherichia+coli%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">A trapping approach reveals novel substrates and physiological functions of the essential protease ftsh in Escherichia coli.</a> J Biol Chem. 287 (51), 42962-42971 (2012).</li>
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<li>Ogura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Balanced+biosynthesis+of+major+membrane+components+through+regulated+degradation+of+the+committed+enzyme+of+lipid+a+biosynthesis+by+the+AAA+protease+FtsH+%28HflB%29+in+Escherichia+coli%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid a biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli.</a> Mol Microbiol. 31 (3), 833-844 (1999).</li>
<li>Klein, G., Kobylak, N., Lindner, B., Stupak, A., Raina, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+of+lipopolysaccharide+in+Escherichia+coli+requires+the+essential+LapB+heat+shock+protein%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein.</a> J Biol Chem. 289 (21), 14829-14853 (2014).</li>
<li>Mahalakshmi, S., Sunayana, M. R., Saisree, L., Reddy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((YciM+is+an+essential+gene+required+for+regulation+of+lipopolysaccharide+synthesis+in+Escherichia+coli%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">YciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli.</a> Mol Microbiol. 91 (1), 145-157 (2014).</li>
<li>Nicolaes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+the+function+of+YciM%2C+a+heat+shock+membrane+protein+required+to+maintain+envelope+integrity+in+Escherichia+coli%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.</a> J Bacteriol. 196 (2), 300-309 (2014).</li>
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<li>Yang, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding-degradation+relationship+of+a+membrane+protein+mediated+by+the+universally+conserved+ATP-dependent+protease+FtsH%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Folding-degradation relationship of a membrane protein mediated by the universally conserved ATP-dependent protease FtsH.</a> J Am Chem Soc. 140 (13), 4656-4665 (2018).</li>
<li>Geertsma, E. R., Mahmood, N. A. B. N., Schuurman-Wolters, G. K., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+reconstitution+of+ABC+transporters+and+assays+of+translocator+function%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Membrane reconstitution of ABC transporters and assays of translocator function.</a> Nat Protoc. 3 (2), 256-266 (2008).</li>
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</ol>

&#65279;The authors declare that they have no competing financial interests.

The first critical step of this protocol is Atto488 labeling of LpxC. A moderate labeling is critical for accurately measuring in vitro degradation activity. Based on our optimization experiments, a molar ratio of LpxC to Atto488 between 9:1 and 3:1 is optimal. Excessive Atto488 results in over-labeling, which likely perturbs the native state of LpxC. Under these conditions, the difference in degradation of LpxC&#8211;Atto488 by FtsH alone versus FtsH/LapB is markedly reduced (data not shown). Conversely, insuff...

&#65279; AAA+ proteases are conserved, ATP-dependent molecular machines that play central roles in protein quality control and regulatory proteolysis1,2. These machines contain a hexameric ring of AAA+ ATPases domain, which hydrolyze ATP, recognize, unfold, and &#65279;translocate the substrates to the compartmental protease domain for degradation1. While cytoplasmic AAA+ proteases such as ClpXP3 and Lon4 have been extensively characterized using well-established in vitro assays, membrane-bound AAA+ proteases remain comparatively poorly understood. This gap largely reflects the technical challenges associated with reconstituting membrane proteins into defined lipid environments and quantitatively measuring their proteolytic activity in vitro. The overall goal of this method is to establish sensitive, quantitative assays for investigating the proteolytic activity of membrane-bound proteases.
FtsH is the only essential AAA+ protease in Escherichia coli and serves as a prototypical membrane-bound AAA+ protease5,6,7. Anchored to the inner membrane by N-terminal transmembrane helices, FtsH degrades a diverse set of cytoplasmic and membrane-associated substrates, thereby regulating membrane protein quality control, stress responses, and lipid homeostasis8,9,10,11. One of its best-characterized substrates is LpxC, a soluble deacetylase that catalyzes the first committed step in lipopolysaccharide (LPS) biosynthesis12,13. Efficient degradation of LpxC by FtsH requires the membrane protein LapB, which functions as an adaptor to specifically recognize LpxC and deliver it to the protease14,15,16,17,18.
Despite its physiological importance, quantitative analysis of FtsH-mediated proteolysis in vitro has been limited, particularly for cytoplasmic substrates such as LpxC. Traditional in vitro degradation assays use FtsH purified in detergent micelles13,19, which do not provide the membrane environment for the membrane-anchored protease. In addition, the degradation assays often rely on SDS&#8211;PAGE&#8211;based detection of substrate disappearance13, which can be labor-intensive, low-throughput, and insufficiently sensitive for kinetic analyses. More recently, FtsH reconstituted into bicelles has been used to study degradation of membrane substrates, as pioneered by Heedeok Hong&#8217;s group20; however, a comparable system for analyzing cytoplasmic substrates remains lacking. Here, we adapt Heedeok Hong&#8217;s bicelle reconstitution to proteoliposome reconstitution to mimic the membrane environment. Besides that, the water-soluble substrate LpxC is fluorescence-labeled, which enables sensitive and quantitative measurement of the kinetics of FtsH/LapB-dependent degradation of LpxC in vitro.
This method should be used when researchers need to quantitatively measure the FtsH-mediated proteolysis of water-soluble substrates. It provides several advantages over existing approaches. First, fluorescence-based detection of proteolytic products, rather than substrate disappearance, substantially improves sensitivity and dynamic range, facilitating quantitative comparisons of proteolytic activity. Second, the use of proteoliposomes preserves the membrane context required for FtsH function while allowing precise control over protein and lipid composition18. Finally, although developed using the FtsH&#8211;LapB&#8211;LpxC system, this protocol is readily adaptable to other membrane-bound AAA+ proteases and their cytoplasmic substrates that require membrane-associated degradation machinery.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>USA scientific</td><td>1415-2500</td><td></td></tr><tr><td>200 nm polycarbonate membrane</td><td>Avanti Polar Lipids</td><td>610006</td><td></td></tr><tr><td>4-20% SDS-PAGE</td><td></td><td></td><td>Home made</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma</td><td>A26209</td><td></td></tr><tr><td>Atto 488 Protein Labeling Kit</td><td>Sigma</td><td>38371</td><td></td></tr><tr><td>Bio-Beads SM-2&amp;nbsp;</td><td>Bio-Rad</td><td>152-8920</td><td>Nonpolar polystyrene adsorbent&amp;nbsp;</td></tr><tr><td>Black 96-well plate&amp;nbsp;</td><td>Costar</td><td>3915</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td></td></tr><tr><td>Coomassie Brilliant Blue R250</td><td>Sigma</td><td>1.12553</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Filter support</td><td>Avanti Polar Lipids</td><td>610014</td><td></td></tr><tr><td>Glass syringe</td><td>Avanti Polar Lipids</td><td>610017</td><td></td></tr><tr><td>Glass test tube</td><td>KIMBLE</td><td>45066-16150</td><td></td></tr><tr><td>Glycerol</td><td>Avantor</td><td>2136-01</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate</td><td>Sigma</td><td>228648</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>179337</td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75-002-436</td><td></td></tr><tr><td>Mini-extruder</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>MLA-80 rotor&amp;nbsp;</td><td>Beckman</td><td>367096</td><td>Fixed-angle rotor&amp;nbsp;</td></tr><tr><td>Plate reader</td><td>Tecan</td><td>Infinite&amp;nbsp;M1000 PRO</td><td></td></tr><tr><td>POPC&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>850457C</td><td></td></tr><tr><td>Refrigerated microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75002559</td><td></td></tr><tr><td>SDS</td><td>American Bio</td><td>AB01920-00500</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Sodium dihydrogen phosphate</td><td>Sigma</td><td>38400100</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>567547</td><td></td></tr><tr><td>Sonicator</td><td>Fisherbrand</td><td>FB-11201</td><td></td></tr><tr><td>Spectrophotometer</td><td>SCILOGEX</td><td>SCI-V1000</td><td></td></tr><tr><td>Superdex 200 Increase 10/300 GL</td><td>Cytiva&amp;nbsp;</td><td>28990944</td><td>High-resolution gel filtration column&amp;nbsp;</td></tr><tr><td>Tabletop ultracentrifuge</td><td>Beckman</td><td>Optima MAX-XP</td><td></td></tr><tr><td>TCEP HCl</td><td>Gold Biotechnology</td><td>TCEP1</td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>Sigma</td><td>T6399</td><td></td></tr><tr><td>Tris Base</td><td>Sigma</td><td>252859</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>355647</td><td></td></tr><tr><td>Ultrafiltration column&amp;nbsp;</td><td>Millipore</td><td>UFC8030</td><td></td></tr><tr><td>Vortex-Genie 2 Mixer</td><td>Scientific Industries</td><td>SI-0236&amp;nbsp;</td><td></td></tr><tr><td>Zinc acetate dihydrate</td><td>Sigma</td><td>379786</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>444203</td><td></td></tr></tbody></table>

an in vitro assay for lpxc degradation by the membrane-bound protease ftsh and adaptor lapb from escherichia coli

An In Vitro Assay For LpxC Degradation By The Membrane-Bound Protease FtsH And Adaptor LapB From Escherichia coli

This protocol presents a sensitive, quantitative in vitro degradation assay of LpxC using reconstituted AAA+ (ATPase associated with diverse cellular activities) protease FtsH and adaptor LapB in proteoliposomes. AAA+ proteases are ATP-dependent molecular machines that maintain protein homeostasis and regulate diverse cellular processes in the cytoplasm or at cellular membranes. Compared with their cytoplasmic counterparts, membrane-bound AAA+ proteases remain poorly characterized due to technical challenges in reconstituting and measuring their activity in vitro. Here, we use the FtsH&#8211;LapB&#8211;LpxC system as a model to develop a fluorescence-based degradation assay in proteoliposomes. FtsH and LapB are reconstituted into proteoliposomes to mimic the native membrane environment, and LpxC is covalently labeled with the fluorescent dye Atto488. Degradation is initiated by combining proteoliposomes with labeled LpxC and ATP and terminated by trichloroacetic acid (TCA). A successful assay is defined by a time-dependent increase in fluorescence in the soluble supernatant fraction after TCA precipitation, corresponding to the release of fluorescent peptide fragments generated by LpxC proteolysis. This signal enables direct quantification of initial degradation rates under defined conditions. Compared with traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-based assays, this method provides substantially improved sensitivity and enables quantitative kinetic analysis. Moreover, the proteoliposome system allows systematic investigation of how protein components, such as adaptors and anti-adaptors, as well as lipid composition, influence LpxC degradation. Although developed for the FtsH&#8211;LapB&#8211;LpxC system, this assay is readily adaptable to other membrane-bound AAA+ proteases and substrates, providing a general platform for studying membrane-associated proteolysis in vitro.

To improve the sensitivity of the in vitro degradation assay, the substrate LpxC was labeled with the fluorescent dye Atto488. The succinimidyl ester group of Atto488 reacts efficiently with primary amino groups of LpxC under mildly alkaline conditions. Because Tris buffer contains primary amines that interfere with this reaction, LpxC purified in Tris buffer was exchanged into phosphate buffer by gel filtration before labeling. For the labeling reaction, a molar ratio of LpxC to Atto488 of 3:1 was used to achie...

Escherichia coli regulates lipopolysaccharide (LPS) biosynthesis primarily by controlling the degradation of LpxC, the deacetylase that catalyzes the first committed step of LPS synthesis. The goal of this protocol is to establish a sensitive, quantitative in vitro degradation assay for LpxC, using reconstituted AAA+ protease FtsH and adaptor LapB in proteoliposomes.

This protocol presents a sensitive, quantitative in vitro degradation assay of LpxC using reconstituted AAA+ (ATPase associated with diverse cellular ...

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Learn a fluorescence-based in vitro assay for LpxC degradation by the membrane-bound protease FtsH and adaptor LapB from Escherichia coli. This sensitive method enables quantitative kinetic analysis of membrane-associated proteolysis.

An In Vitro Assay For LpxC Degradation By The Membrane-Bound Protease FtsH And Adaptor LapB From Escherichia coli

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