Proteolysis mediated by the membrane-integrated ATP-dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates

Abstract

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane-integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40-60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water-soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.

Keywords: AAA+ protease; ATP hydrolysis rate; FtsH; cooperativity; membrane protein degradation; membrane protein folding; membrane protein quality control; steric trapping.

Figure 1

The membrane‐integrated AAA+ protease FtsH and the model membrane substrate GlpG. (a) The domain structure and membrane topology of FtsH. TM: Transmembrane segment; PM: periplasmic domain. (b) The GlpG constructs with the C‐ and N‐terminal degradation markers employed in this study. GlpG TM: The transmembrane domain of GlpG (residues 87–276); the 108‐tag: –SLLWS; the YccA_N tag: MDRIVSSSHDRTSLLSTHKVLRN‐. (c) Reconstitution of FtsH‐mediated degradation of GlpG in the negatively charged DMPC/DMPG/CHAPS bicelles (molar ratio, 3:1:1). The thiol‐reactive fluorescent NBD label as a reporter group to monitor degradation was conjugated to the cytoplasmic interfacial region of the middle helix TM3 of the GlpG variant G172C (2IC8) (Reference 49). The structure of the cytoplasmic portion of FtsH from Thermotoga maritima (2CEA) is employed. (Reference 8).

Figure 2

FtsH‐mediated degradation of GlpG in bicelles at various ATP concentrations. (Left) Time‐dependent degradation monitored by NBD fluorescence. (Right) End‐point degradation measured by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) after 16 h. Top: Degradation of GlpG‐108; middle: degradation of YccA_N‐GlpG; and bottom: degradation of the destabilized variant M100A of GlpG‐108. All assays were performed at 37°C with FtsH (2 μM) and GlpG (15 μM) in DMPC/DMPG/CHAPS bicelles (pH 7.5).

Figure 3

Dependence of the degradation rates of the membrane protein GlpG and water‐soluble casein on the ATP hydrolysis rates. (a) (Left) degradation rates of GlpG‐108, YccA_N‐GlpG and GlpG‐108 M100A as a function of ATP hydrolysis rate at 37°C. The positions of the maximal ATP hydrolysis rates (k _cat,ATP) and the half maxima (k _cat,ATP/2) are marked with block arrows. The threshold ATP hydrolysis rates are marked with line arrows. (Right) The effective ATP cost for degradation of each GlpG variant (in parenthesis) was obtained from the inverse of the maximum value in the first derivative of the plot in left. (b) (Left) Time‐dependent degradation of water‐soluble Bodipy FL‐labeled casein (0.45 mg/mL) by FtsH (2 μM) at various ATP concentrations in neutral DMPC/CHAPS bicelles (molar ratio, 2.5:1). Degradation was monitored by dequenching of Bodipy FL fluorescence (λ _excitation = 485 nm; λ _emission = 525 nm) at 37°C. (Right) Dependence of the degradation rates of Bodipy FL casein on the ATP hydrolysis rates in comparison to that of GlpG‐108. Values are means ± SEM (N = 2–5). (c) (Left) Time‐dependent degradation of Bodipy FL‐labeled casein (0.08 mg/mL) by E. coli Lon (2 μM) in 20 mM HEPES (pH 7.5), 100 mM KCl, and 1 mM dithiothreitol (DTT) at 30°C. (Right) The relationship between degradation and ATP hydrolysis rates mediated by Lon.

Figure 4

The effects of the macromolecular crowding agent Ficoll 70 on ATPase activity of FtsH and thermodynamic stability of GlpG. (a) Dependence of ATP hydrolysis rates of FtsH (2 μM) on the concentration of Ficoll 70 measured at a saturating concentration of ATP (5 mM) in DMPC/DMPG/CHAPS bicelles at 37°C (see also Fig. S6). (b) Michaelis–Menten analysis of ATPase activity in DMPC/DMPG/CHAPS bicelles in the presence and absence of 15% Ficoll 70 (w/v). (c) The principle of steric trapping.52 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 the intrinsic binding affinity (ΔG ^o _Bind). Because of steric hindrance, the second mSA binds only when native tertiary contacts are unraveled by transient unfolding. Hence, binding of the second mSA is attenuated depending on the stability of the target protein (ΔG ^o _Bind + ΔG ^o _U). By adjusting the biotin affinity of mSA by mutation, unfolding and binding reactions can be reversibly controlled, and ΔG ^o _U of the target protein can be obtained by monitoring the binding of the second mSA. Binding of mSA to biotin labels on GlpG was measured by FRET‐based assay employing the BtnPyr label (donor) and mSA labeled with a nonfluorescent dabcyl quencher (acceptor) [Ref. 52 Fig. S7(a)]. (d) The effect of Ficoll 70 on the stability of GlpG in dodecylmaltoside detergent (5 mM). Binding isotherms between double‐biotin variants of GlpG (172/267‐BtnPyr₂) and a mSA_DAB variant with a reduced biotin‐binding affinity (mSA_DAB‐S27A, K _d,biotin = 1.4 nM) monitored by quenching of pyrene fluorescence.52 15% Ficoll 70 does not affect the intrinsic biotin‐binding affinity of mSA to the biotin labels [Fig. S7(B)]. Values are means ± SEM (N = 2–3).

Figure 5

The effect of the macromolecular crowding agent Ficoll 70 on the relationship between the degradation and ATP hydrolysis rates. (a) Degradation rates of GlpG‐108 in DMPC/DMPG/CHAPS bicelles measured as a function of ATP hydrolysis rate in the presence of 15% Ficoll 70. The data were compared with those in the absence of Ficoll 70. Values are means ± SEM (N = 2). (b) The influence of Ficoll 70 on the effective ATP cost (parenthesis) obtained from the inverse of the first derivative of the plot in Figure 5(a).