Abstract
AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.
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References
Langklotz S, Narberhaus F (2011) The Escherichia coli replication inhibitor CspD is subject to growth-regulated degradation by the Lon protease. Mol Microbiol 80(5):1313–1325
Schäkermann M, Langklotz S, Narberhaus F (2013) FtsH-mediated coordination of lipopolysaccharide biosynthesis in Escherichia coli correlates with the growth rate and the alarmone (p)ppGpp. J Bacteriol 195(9):1912–1919
Tatsuta T, Tomoyasu T, Bukau B, Kitagawa M, Mori H, Karata K, Ogura T (1998) Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo. Mol Microbiol 30(3):583–593
Sauer RT, Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587–612
Battesti A, Gottesman S (2013) Roles of adaptor proteins in regulation of bacterial proteolysis. Curr Opin Microbiol 16(2):140–147
Gur E, Biran D, Ron EZ (2011) Regulated proteolysis in Gram-negative bacteria--how and when? Nat Rev Microbiol 9(12):839–848
Konovalova A, Søgaard-Andersen L, Kroos L (2014) Regulated proteolysis in bacterial development. FEMS Microbiol Rev 38(3):493–522
Langklotz S, Baumann U, Narberhaus F (2012) Structure and function of the bacterial AAA protease FtsH. Biochim Biophys Acta 1823(1):40–48
Ogura T, Inoue K, Tatsuta T, Suzaki T, Karata K, Young K, Su LH, Fierke CA, Jackman JE, Raetz CR, Coleman J, Tomoyasu T, Matsuzawa H (1999) Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli. Mol Microbiol 31(3):833–844
Führer F, Langklotz S, Narberhaus F (2006) The C-terminal end of LpxC is required for degradation by the FtsH protease. Mol Microbiol 59(3):1025–1036
Flynn JM, Neher SB, Kim YI, Sauer RT, Baker TA (2003) Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol Cell 11(3):671–683
Feng J, Michalik S, Varming AN, Andersen JH, Albrecht D, Jelsbak L, Krieger S, Ohlsen K, Hecker M, Gerth U, Ingmer H, Frees D (2013) Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus. J Proteome Res 12(2):547–558
Bhat NH, Vass RH, Stoddard PR, Shin DK, Chien P (2013) Identification of ClpP substrates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development. Mol Microbiol 88(6):1083–1092
Westphal K, Langklotz S, Thomanek N, Narberhaus F (2012) A trapping approach reveals novel substrates and physiological functions of the essential protease FtsH in Escherichia coli. J Biol Chem 287(51):42962–42971
Kirstein J, Hoffmann A, Lilie H, Schmidt R, Rübsamen-Waigmann H, Brötz-Oesterhelt H, Mogk A, Turgay K (2009) The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease. EMBO Mol Med 1(1):37–49
Kihara A, Akiyama Y, Ito K (1995) FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit. Proc Natl Acad Sci U S A 92(10):4532–4536
Bertani D, Oppenheim AB, Narberhaus F (2001) An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease. FEBS Lett 493(1):17–20
Katz C, Ron EZ (2008) Dual role of FtsH in regulating lipopolysaccharide biosynthesis in Escherichia coli. J Bacteriol 190(21):7117–7122
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372
Megger DA, Bracht T, Meyer HE, Sitek B (2013) Label-free quantification in clinical proteomics. Biochim Biophys Acta 1834(8):1581–1590
Acknowledgments
The authors would like to thank Alexandra Müller, Sina Langklotz, and Thilo Lerari for carefully reading the manuscript and for many helpful comments. The work was supported by grants from the German Research Foundation (DFG, SFB642: GTP- and ATP-dependent membrane processes) and P.U.R.E. (Protein Unit for Research in Europe, funded by the German federal state North Rhine-Westphalia).
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Lindemann, C., Thomanek, N., Kuhlmann, K., Meyer, H.E., Marcus, K., Narberhaus, F. (2018). Next-Generation Trapping of Protease Substrates by Label-Free Proteomics. In: Becher, D. (eds) Microbial Proteomics. Methods in Molecular Biology, vol 1841. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8695-8_14
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DOI: https://doi.org/10.1007/978-1-4939-8695-8_14
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