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Generation of Artificial N-end Rule Substrate Proteins In Vivo and In Vitro

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Plant Proteostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1450))

Abstract

In order to determine the stability of a protein or protein fragment dependent on its N-terminal amino acid, and therefore relate its half-life to the N-end rule pathway of targeted protein degradation (NERD), non-Methionine (Met) amino acids need to be exposed at their amino terminal in most cases. Per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. Moreover, almost exclusively, stabilizing, or not per se destabilizing residues are N-terminally exposed upon Met excision by Met aminopeptidases. To date, there exist two prominent protocols to study the impact of destabilizing residues at the N-terminal of a given protein by selectively exposing the amino acid residue to be tested. Such proteins can be used to study NERD substrate candidates and analyze NERD enzymatic components. Namely, the well-established ubiquitin fusion technique (UFT) is used in vivo or in cell-free transcription/translation systems in vitro to produce a desired N‐terminal residue in a protein of interest, whereas the proteolytic cleavage of recombinant fusion proteins by tobacco etch virus (TEV) protease is used in vitro to purify proteins with distinct N-termini. Here, we discuss how to accomplish in vivo and in vitro expression and modification of NERD substrate proteins that may be used as stability tester or activity reporter proteins and to characterize potential NERD substrates.

The methods to generate artificial substrates via UFT or TEV cleavage are described here and can be used either in vivo in the context of stably transformed plants and cell culture expressing chimeric constructs or in vitro in cell-free systems such as rabbit reticulocyte lysate as well as after expression and purification of recombinant proteins from various hosts.

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Acknowledgments

The authors thank Carolin Mai for critical reading and helpful comments on the manuscript and Andreas Bachmair for sharing pRTUB8 containing the plant codon-optimized synthetic human Ub gene. This work was supported by a grant for setting up the junior research group of the ScienceCampus Halle—Plant-based Bioeconomy to N.D., by a Ph.D. fellowship of the Landesgraduiertenförderung Sachsen-Anhalt awarded to C.N., and a grant of the Leibniz-DAAD Research Fellowship Programme by the Leibniz Association and the German Academic Exchange Service (DAAD) to A.C.M. and N.D. Financial support came from the Leibniz Association, the state of Saxony Anhalt, the Deutsche Forschungsgemeinschaft (DFG) Graduate Training Center GRK1026 “Conformational Transitions in Macromolecular Interactions” at Halle, and the Leibniz Institute of Plant Biochemistry (IPB) at Halle, Germany. To complete work on this project, a Short Term Scientific Mission (STSM) of the European Cooperation in Science and Technology (COST) was granted to A.C.M. and N.D. by the COST Action BM1307—“European network to integrate research on intracellular proteolysis pathways in health and disease (PROTEOSTASIS)”.

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Authors and Affiliations

  1. Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany

    Christin Naumann, Augustin C. Mot & Nico Dissmeyer

  2. ScienceCampus Halle – Plant-Based Bioeconomy, Halle (Saale), Germany

    Christin Naumann, Augustin C. Mot & Nico Dissmeyer

  3. Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Cluj-Napoca, Romania

    Augustin C. Mot

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  1. Christin Naumann
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  2. Augustin C. Mot
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  3. Nico Dissmeyer
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Correspondence to Nico Dissmeyer .

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  1. Development Program, CRAG (CSIC-IRTA-UAB-UB) Edifici CRAGCampus, UAB Bellaterra (Cerdanyola del Vallés), Barcelona, Spain

    L. Maria Lois

  2. Computational and Experimental Biology Group, National Health Institute Doctor Ricardo Jorge, IP, Lisbon, Portugal

    Rune Matthiesen

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Naumann, C., Mot, A.C., Dissmeyer, N. (2016). Generation of Artificial N-end Rule Substrate Proteins In Vivo and In Vitro. In: Lois, L., Matthiesen, R. (eds) Plant Proteostasis. Methods in Molecular Biology, vol 1450. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3759-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-3759-2_6

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