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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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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