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

Summary

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.

Abstract

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–LapB–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–LapB–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.

Introduction

 AAA+ proteases are conserved, ATP-dependent molecular machines that play central roles in protein quality control and regulatory proteolysis^1,2. These machines contain a hexameric ring of AAA+ ATPases domain, which hydrolyze ATP, recognize, unfold, and 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+ protease^5,6,7. 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 homeostasis^8,9,10,11. One of its best-characterized substrates is LpxC, a soluble deacetylase that catalyzes the first committed step in lipopolysaccharide (LPS) biosynthesis^12,13. 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 protease^{14,15,16,17,18}.

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 micelles^13,19, which do not provide the membrane environment for the membrane-anchored protease. In addition, the degradation assays often rely on SDS–PAGE–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’s group²⁰; however, a comparable system for analyzing cytoplasmic substrates remains lacking. Here, we adapt Heedeok Hong’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–LapB–LpxC system, this protocol is readily adaptable to other membrane-bound AAA+ proteases and their cytoplasmic substrates that require membrane-associated degradation machinery.

Protocol

1. Labeling LpxC with Atto488

1. LpxC preparation
   1. 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).
   2. Concentrate LpxC using an ultrafiltration column to a final concentration of at least 5 mg/mL at 4 °C.
2. Labeling LpxC with Atto488
   1. Dilute 0.5 mg of LpxC in 50 mM phosphate buffer, pH 7.6, to a final volume of 225 µL. Add 25 µL of 1 M sodium bicarbonate to adjust the pH to approximately 8.3.
   2. Dissolve 0.24 mg of Atto488 reactive dye in 20 µL of 0.1 M sodium bicarbonate buffer to prepare a stock solution (0.012 mg/µL). Prepare a working solution by diluting 2 µL of the stock solution with 18 µL of 0.1 M sodium bicarbonate buffer, yielding a final concentration of 0.0012 mg/µL.
   3. Immediately add 4 µL 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 °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 °C.
3. Separation of LpxC–Atto488 from free dye
   1. Equilibrate a PD-10 desalting column with 10 mL of ultrapure water, followed by 15 mL of 50 mM phosphate buffer, pH 7.6.
   2. Apply the labeling reaction mixture to the column and allow it to fully enter the resin.
   3. Elute with 5 mL of 50 mM phosphate buffer, pH 7.6. Two distinct bands corresponding to LpxC–Atto488 (first band) and free Atto488 dye (second band) should become visible. Collect fractions from the first band in ~0.5 mL aliquots.
   4. Analyze eluted fractions by SDS-PAGE. Visualize fluorescence under UV illumination before Coomassie Brilliant Blue (CBB) staining.
   5. Pool fractions containing LpxC–Atto488 and concentrate the labeled protein to approximately 2 mg/mL.
   6. Estimate labeling efficiency by measuring the protein concentration and Atto488 concentration using a microvolume spectrophotometer.
      NOTE: A ratio of Atto488 concentration (in pmol/µL) to LpxC concentration (in mg/mL) in the range of 2–5 indicates an appropriate labeling level. This corresponds to a molar ratio of Atto488 to LpxC ranging from 1:15 to 1:6 after labeling.
   7. Aliquot LpxC–Atto488 into 1.5 mL microcentrifuge tubes, flash-freeze in liquid nitrogen, and store at -80 °C. 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).

2. Reconstitution of FtsH/LapB into proteoliposomes

1. Liposomes preparation
   1. 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 N₂, followed by overnight drying under vacuum.
      NOTE: POPC has a phase transition temperature of ~-2 °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.
   2. Add 1,315 µL 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 °C. At this point, the protocol can be paused for up to 1 month.
   3. Aliquot the liposome suspension into 200 µL portions and store at -20 °C.
2. Liposomes extrusion
   1. Thaw a 200 µL liposome aliquot in a water bath at room temperature and dilute with 800 µL of buffer A to a final volume of 1 mL.
   2. Subject the liposomes to three freeze-thaw cycles by alternating between liquid nitrogen and a water bath at room temperature.
   3. 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.
   4. Divide the 1 mL extruded liposome suspension into three tubes (330 µL per tube) and dilute each with 600 µL of buffer A.
3. Triton X-100 swelling titration
   NOTE: Triton X-100 is a low critical micelle concentration (CMC) detergent, and 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.
   1. Prepare 10% (v/v) Triton X-100 by mixing 100 µL Triton X-100 with 900 µL ultrapure water, and incubating it in a shaker to fully dissolve Triton X-100.
   2. Measure the absorbance of liposomes at 540 nm (A₅₄₀) using a spectrophotometer.
   3. Add 5 µL aliquots of 10% Triton X-100 sequentially, gently mixing after each addition, and measure A₅₄₀. Continue adding Triton X-100 until A₅₄₀ reaches a maximum, then add Triton X-100 an additional 4–5 times until the absorbance decreases to approximately 60% of the peak value.
   4. Based on the titration curve, swell liposomes by adding 35 µL of 10% Triton X-100 and incubate at room temperature for 1 h with gentle agitation.
4. Reconstitution of FtsH/LapB into liposomes
   1. Express and purify FtsH and LapB proteins as described previously¹⁸.
   2. 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 °C.
   3. For each tube of Triton X-100–swollen liposomes, add 3 µL of 500 mM tris(2-carboxyethyl)phosphine (TCEP), 22.5 µg of FtsH, and 22.5 µg of LapB. Adjust the total volume to 1 mL with buffer A and incubate at RT for 1 h with gentle agitation.
      NOTE: Protein-to-lipid ratios of 1:20 to 1:200 (wt/wt) were routinely used during proteoliposomes reconstitution²¹. Here, a ratio of 1:100 is used.
   4. 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 °C overnight, and finally 40 mg at 4 °C for 1 h.
   5. Briefly centrifuge to pellet the nonpolar polystyrene adsorbent at 4 °C (400 × 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.
   6. Balance the tubes and ultracentrifuge at 250,000 × g at 4 °C for 45 min in a fixed-angle rotor (e.g., MLA-80) to pellet proteoliposomes.
   7. Discard the supernatant and resuspend the proteoliposome pellet in 70 µL of buffer A supplemented with 1 mM TCEP.
      NOTE: The final proteoliposome volume is approximately 100 µL.
   8. Flash-freeze the proteoliposomes in liquid nitrogen and store at -80 °C. 
      NOTE: At this point, the protocol can be paused for up to 1 month.
5. SDS-PAGE analysis of proteoliposomes
   1. Mix proteoliposomes thoroughly and combine 5 µL of the sample with 5 µL of 2× SDS loading buffer (0.1 M Tris-HCl, pH 6.8, 5% SDS, 20% glycerol, 0.01% bromophenol blue, 200 mM DTT).
   2. Load proteoliposome samples alongside 525 ng of FtsH (corresponding to 0.06 µM FtsH hexamer in 20 µL) on a 4–20% SDS-PAGE gel.
   3. Stain the gel with CBB.
   4. 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.

3. In vitro degradation of LpxC–Atto488

1. Perform in vitro degradation assays in a 20 µL reaction containing FtsH/LapB proteoliposomes (corresponding to 0.06 µM FtsH) and 0.5, 1, 2, 2.5, 5, 10, 20, 40 µM LpxC-Atto488 in degradation buffer (50 mM Tris-acetate, pH 8.0, 10 mM Mg-acetate, 5 mM ATP, 25 µM Zn-acetate, 80 mM NaCl, 1.4 mM β-mercaptoethanol).
2. For the measurement of each concentration of substrate, prepare a reaction of 70 µL.
   1. Take a 20 µL fraction and quench it by immediately mixing it with 48 µL 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.
   2. 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–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).
3. Incubate samples at 37 °C for 30 min to precipitate proteins, then centrifuge at 18,800 × g at room temperature for 20 min.
4. Neutralize samples by transferring 50 µL of supernatant into a tube containing 60 µL of 500 mM Tris-HCl, pH 8.8. Mix well and transfer 100 µL to a black 96-well plate. Measure fluorescence using a plate reader with excitation at 498 nm and emission at 520 nm.

Results

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...

Discussion

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–Atto488 by FtsH alone versus FtsH/LapB is markedly reduced (data not shown). Conversely, insuff...

Materials

List of materials used in this article

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