Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

Abstract

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380-550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.

Figure 1

FtsH as a model degradation machine and bicelles as a model lipid medium. (a) Domain architecture and topology of FtsH in the E. coli inner membrane. TM: trans-membrane domain; PM: periplasmic domain. (b) Comparison of intrinsic ATPase activity of FtsH in 3% (w/v) Triton X- 100, bicelles (neutral DMPC/CHAPS, molar ratio = 2.8:1; negatively charged DMPC/DMPG/CHAPS, molar ratio = 3:1:1.4), liposomes (E. coli phospholipids; DMPC/DMPG, molar ratio = 3:1) and CHAPS. The data were fitted to Michaelis-Menten (solid lines) and Hill (dashed lines) equations (see Supporting Methods). Data are represented as mean ± SEM (n = 3). (c) Diffusion of NBD- and rhodamine-labeled (top) lipids and (bottom) GlpG in 3% DMPC/DMPG/CHAPS bicelles at 37°C monitored by FRET, i.e., the intensity ratio of NBD fluorescence at 535 nm to rhodmaine fluorescence at 595 nm. Dead time of mixing was ∼15 sec. (Right) NBD and rhodamine fluorescence spectra after the end of each mixing reaction.

Figure 2

Degradation of GlpG variants in vivo. (a) Structure of the TM domain of GlpG (GlpG TM: residues 87–276, PDB code: 3B45³³) with the topology in the E. coli inner membrane. (b–c) (Left) GlpG constructs for testing the effect of the C- or N-terminal degradation markers on GlpG degradation. (Right) Time-dependent degradation of GlpG variants monitored by Western blotting in the E. coli −ftsH and +ftsH strains. Time 0 indicates the time when spectinomycin was added to block protein synthesis.

Figure 3

GlpG degradation by FtsH in bicelles. (a) Principle of the fluorescence-based degradation assay for measuring degradation rates of GlpG in vitro. GlpG with an engineered Cys residue (G172C) was labeled with thiol-reactive environment-sensitive fluorophore NBD. Upon degradation, NBD fluorescence is quenched due to the transfer of NBD label from the bicellar phase to the aqueous phase. (b) (Left) Time-dependent degradation of GlpG variants (5 mM) by FtsH ([FtsH₆] = 0.5 mM) monitored by NBD fluorescence in 3% DMPC/DMPG/CHAPS bicelles at 37°C. Fluorescence intensity with 5 mM ATP at each time point was normalized to the intensity without ATP. (Right) Degradation of GlpG variants monitored by SDS-PAGE and Coomassie-blue staining. (c) Comparison of degradation of GlpG-108 monitored by NBD fluorescence and SDS-PAGE shown in (b). (d) Kinetic analysis of FtsH-mediated degradation of GlpG with various degradation markers. Data are represented as mean ± SEM (n = 3).

Figure 4

Steric trapping to measure spontaneous unfolding rate of GlpG. (a) Principle of steric trapping. When biotin tags are conjugated to two specific residues that are spatially close in the folded state but distant in the amino acid sequence, the first mSA binds either biotin label with intrinsic binding affinity, but due to steric hindrance, the second mSA binds only when native tertiary contacts are unraveled by transient unfolding. k_on: on-rate constant of mSA binding to biotin label; k_off: off-rate constant of mSA-biotin complex; k_U: spontaneous unfolding rate; k_F: refolding rate. (b) Unfolding kinetics of double biotin variants of GlpG measured by steric trapping in 3% DMPC/DMPG/CHAPS bicelles at 37°C. The N- and C-subdomains were color-coded in cyan and orange, respectively. GlpG activity with mSA relative to that without mSA was used as an unfolding readout. Errors designate ± STD from fitting.

Figure 5

The conformational stability and hydrophobicity of GlpG control degradation. (a) The residues for amino acid substitutions to decrease the conformational stability (M100A) or increase the hydrophobicity of the C-terminal TM6 helix (A259L/A263L/V267W/L270F, designated as “LLWF”). (b) (Left) Arrhenius plot for measuring the activation energy of unfolding (E_a,U) of GlpG WT and M100A variant. Spontaneous unfolding rates (k_U) were measured using steric trapping in DDM at various temperatures. (Right) Unfolding energy landscape of GlpG WT and M100A variant in DDM including the thermodynamic stability (ΔG_U) and E_a,U. F, U and TS denote the folded state, unfolded state and transition state, respectively. (c) The effect of M100A substitution on k_U of GlpG measured by steric trapping in 3% DMPC/DMPG/CHAPS bicelles at 37°C. Errors designate ± STD from fitting. (d) The effect of M100A substitution on the degradation rates of GlpG with the C-terminal (the 108 tag) or N-terminal (the YccA_N tag) degradation marker. Data are represented as mean ± SEM (n = 3–6). (e) The whole-residue hydropathy plot of the GlpG TM domain, and M100A and LLWF variants using Hessa-von Heijne (HvH), Wimley-White (WW) octanol and Tian-Lin-Liang (TLL) depth-dependent hydrophobicity scales. (f) The effect of LLWF substitution on the degradation rate of GlpG with the 108 tag. The degradation rates of GlpG_WT-108 and YccA_N-GlpG_WT were also plotted for comparison. Data are represented as mean ± SEM (n = 3).

Figure 6

Three-step model of FtsH-mediated degradation of membrane proteins.