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 ....

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 bu....

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