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, Abbreviations: 108, an amino acid sequence, SLLWS; AAA+, ATPases associated with diverse cellular activities; ATP, adenosine triphosphate; E. coli, Escherichia coli; E a,U , activation energy of unfolding; GFP, green fluorescent protein; mSA, monovalent streptavidin; NBD, nitrobenzoxadiazole; n H , the Hill coefficient; PAN, proteasome-activating AAA+ nucleotidase; TM, transmembrane; YccA N , the N-terminal tail of an E. coli membrane protein YccA; ΔG o U , free energy of unfolding. Additional Supporting Information may be found in the online version of this article.Significance statement: FtsH is a membrane-integrated ATP-dependent protease, playing a key role in membrane protein quality control. Here, we investigated how FtsH utilizes ATP hydrolysis to degrade proteins. We find that FtsH couples multiple ATP hydrolysis events to degradation in a highly cooperative and efficient manner. This mechanism explains how FtsH overcomes large energetic costs in unfolding substrates in the membranes and extracting them toward its protease domain located outside the membrane.which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.

FtsH degrades dihydrofolate reductase by recognizing a partially folded species

“…Although we find that FtsH does degrade DHFR, our results suggest that the actual target may be a less stable intermediate in folding/unfolding, leaving the question of unfolding strength open. The finding that FtsH can extract and degrade the stable GlpG protein from the membrane provides the strongest evidence that FtsH possesses a robust unfolding activity 18,19 . In general, however, we note that AAA+ proteases and substrates co‐evolve to allow the degradation of appropriate cellular proteins when necessary.…”

“…Differences in how FtsH or the substrates were purified might also account for the discrepancy between our results and the previous report. Although the steady-state degradation parameters are similar for ssrA-tagged and untagged DHFR variants, untagged DHFR and DHFR-ssrA 11 have slightly weaker K M and slightly higher k deg values compared to DHFR-ssrA 19 and DHFR-ssrA 40 . These small differences probably reflect the fact that degradation of the ssrA-tagged substrates can initiate at the ssrA tag or within DHFR, potentially altering binding and/or how force is applied to unfold the substrate, 37,38 and the fact that fewer amino acids need to be translocated to complete degradation of the shorter substrates.…”

“…Substrates typically alter the rate of ATP hydrolysis by AAA+ proteases. 26 Indeed, near-saturating concentrations of untagged DHFR, DHFR-ssrA 11 , DHFR-ssrA 19 , or DHFR-ssrA 40 increased the rate of ATP hydrolysis by FtsH by 10-20% (Figure 4). Although this small increase may not be mechanistically important, these results support our model that untagged DHFR and the ssrA-tagged variants are being engaged, unfolded, and degraded by FtsH.…”

“…Next, we tested the possibility that FtsH might degrade the ssrA-tailed and untagged DHFR substrates with substantially different steady-state kinetic parameters. For these experiments, we labeled the only solventexposed cysteine residue of DHFR, Cys 152 , with a maleimide-fluorophore and used time-dependent increase in acid-soluble fluorescent peptides as an assay for the rate of FtsH degradation of DHFR-ssrA 40 , DHFR-ssrA 19 , DHFR-ssrA 11 , and untagged DHFR. Figure 3b shows the linear time-dependent increase in acid-soluble fluorescent peptides reflecting FtsH degradation of 16 μM DHFR-ssrA 11 .…”

“…The C-terminal tail (linker/degron) sequences of DHFR-ssrA 19 , DHFR-ssrA 25 , DHFR-ssrA 40 , and DHFR-ssrA 60 were GSH 6 AANDENYALAA, GSHLGLTSH 6 AAN-DENYALAA, GSHLGLIEVEKPLYGVEPFVGTSH 6 AANDENYALAA, and GSHLGLIEVEKPLYGVEPFVGETAHFEIEL-SEPDVHGQWKLTSH 6 AANDENYALAA, respectively ( V13P titin I27 sequences underlined; H 6 sequence; ssrA-tag sequence in italics). DHFR-ssrA 19 , DHFR-ssrA 25 , DHFR-ssrA 40 , and DHFR-ssrA 60 were purified by Ni ++ -NTA affinity chromatography, Source-15Q anion-exchange chromatography, and Superdex-75 size-exclusion chromatography, and stored frozen.…”