Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb;21(2):268-78.
doi: 10.1002/pro.2013. Epub 2012 Jan 4.

Protein unfolding and degradation by the AAA+ Lon protease

Eyal Gur  1 , Marina Vishkautzan, Robert T Sauer
Affiliations

Protein unfolding and degradation by the AAA+ Lon protease

Eyal Gur et al. Protein Sci. 2012 Feb.

Abstract

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Degradation of mDHFR variants by Lon. Protein samples (5 μM) were incubated with Lon protease (0.3 μM hexamer) and ATP (2 mM; plus a regeneration system) at 37°C in the presence or absence of methotrexate (MTX; 10 μM). At different times, aliquots were removed, quenched, and then analyzed by SDS-PAGE and staining with Coomassie blue. Untagged mDHFR was a poor substrate (upper panel) as was mDHFR-sul20C in the presence of MTX (middle panel). In the absence of MTX, mDHFR-sul20C was rapidly degraded (lower panel).
Figure 2
Figure 2
Degradation of C-tagged mDHFR-fusion proteins. A: Degradation of mDHFR-titinI27-sul20C (5 μM) by Lon (2 μM hexamer) in the presence or absence of MTX (10 μM). Pyruvate kinase is part of the ATP-regeneration system. The numbers on the left side of the gel are MR values (kDa) of protein standards. Other conditions were the same as in Figure 1. B: Degradation of the purified mDHFR-tail protein (5 μM) by Lon (0.3 μM hexamer) in the absence of MTX. C: The intensities of the full-length substrate bands in panel A were determined by densitometry, plotted as a function of time, and fit to single-exponential functions (R > 0.999; amplitude 100%) with rate constants of 2.1 × 10−1 min−1 (no MTX) and 2.1 × 10−2 min−1 (plus MTX).
Figure 3
Figure 3
Degradation of N-tagged mDHFR-fusion proteins. A: Degradation of β20-H6-mDHFR (5 μM) by Lon (0.3 μM hexamer) in the presence or absence of MTX (10 μM). Other conditions as in Fig. 1. B: The intensities of the full-length substrate bands in panel A were determined by densitometry, plotted, and fit to single-exponential functions (R > 0.993; amplitude 100%) with rate constants of 8.5 × 10−2 min−1 (no MTX) and 6.5 × 10−2 min−1 (plus MTX). C: The intensities of the tail-mDHFR fragment bands in panel A and in a second experiment with time points of 0, 20, 40, and 60 min (not shown) were determined by densitometry, plotted, and fit to single-exponential functions (R > 0.999). No MTX: rate constant, 8.8 × 10−2 min−1; amplitude, 18%. Plus MTX: rate constant 1.0 × 10−1 min−1; amplitude, 96%.
Figure 4
Figure 4
Degradation of titinI27-fusion proteins. A: The four left lanes show degradation of β-gal3-93-titinI27 (5 μM) by Lon (2 μM hexamer). The four right lanes show degradation of titinI27-β-gal3-93 (5 μM) by Lon (2 μM hexamer). Other conditions were the same as in Figure 1. B: Degradation of titinI27-β-gal3-93 (3 μM) by Lon (6 μM hexamer). The inset shows that the partially degraded fragment initially accumulates and is then degraded. C: Degradation of titinI27-β-gal3-93-titinI27 (3 μM) by Lon (6 μM hexamer). D: Steady-state rates of degradation of different concentrations of 35S-labeled titinI27-fusion variants by Lon (0.1 μM hexamer) were determined by release of acid-soluble peptides. The solid lines are fits to the Hill equation (rate = Vmax × [S]n/(KMn+[S]n). Kinetic parameters are listed in Table I.
Figure 5
Figure 5
Degradation of GFP-fusion proteins by Lon. A: The four left lanes show degradation of β20-titinI27-GFP-H6 (5 μM) by Lon (2 μM hexamer). The four right lanes show degradation of H6-GFP-titinI27-sul20C (5 μM) by Lon (2 μM hexamer). B: Intensities of the full-length substrate bands from panel A were fit to single-exponential functions. For the β20-tagged substrate, the amplitude of the fit was 88% (∼12% of substrates were nondegradable, possibly because they lost the degradation tag) and the rate constant was 0.49 min−1 (R > 0.999). For the sul20C-tagged substrate, the amplitude of the fit was 100% and the rate constant was 0.017 min−1 (R > 0.999). C: The intensities of the partially degraded GFP fragments in panel A were fit to single-exponential functions. For the sul20C-tagged substrate, the amplitude of the fit was 32% and the rate constant was 0.033 min−1 (R > 0.995). For the β20-tagged substrate, the amplitude of the fit was 87.5% and the rate constant was 0.5 min−1 (R > 0.999).
Figure 6
Figure 6
Lon degradation of a disulfide-bonded substrate. A: Nonreducing SDS-PAGE analysis of a protein in which native mDHFR-cys-sul20C was disulfide bonded to unfolded titinI27-CD-cys. The left lane shows the sample after reduction with DTT. The right lane shows the unreduced sample. The numbers on the left side of the gel are MR values (kDa) of protein standards. B: Nonreducing SDS-PAGE of Lon (0.3 μM hexamer) degradation of the disulfide-linked substrate (5 μM) in the presence and absence of MTX. Other conditions as in Figure 1. C: Reducing SDS-PAGE of Lon (0.3 μM hexamer) degradation of the disulfide-linked substrate (5 μM) in the presence and absence of MTX. D: Reducing SDS-PAGE of Lon (0.3 μM hexamer) degradation of a control disulfide-linked substrate lacking the sul20C degradation tag (5 μM) in the presence and absence of MTX.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gottesman S. Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol. 2003;19:565–587. - PubMed
    1. Baker TA, Sauer RT. ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci. 2006;31:647–653. - PMC - PubMed
    1. Schrader EK, Harstad KG, Matouschek A. Targeting proteins for degradation. Nat Chem Biol. 2009;5:815–822. - PMC - PubMed
    1. Sauer RT, Baker TA. AAA+ proteases: ATP-fueled machines of destruction. Annu Rev Biochem. 2011;80:587–612. - PubMed
    1. Koodathingal P, Jaffe NE, Kraut DA, Prakash S, Fishbain S, Herman C, Matouschek A. ATP-dependent proteases differ substantially in their ability to unfold globular proteins. J Biol Chem. 2009;284:18674–18684. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources