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A Quantitative Method to Monitor the Efficacy of Inhibitors Against the Chymotrypsin-Like Activity of the Proteasome in Tobacco Leaf Protoplasts

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Abstract

Proteasome inhibitors are widely used to study the role of the ubiquitin proteasome system (UPS) in various cellular processes. These drugs have been shown to be highly effective in inhibiting the chymotrypsin-like activity of purified Arabidopsis thaliana proteasomes. However, the analysis of their efficacy in vivo is currently hampered by the absence of a simple method for the quantitative determination of proteasomal activity in plant cell extracts. Previous studies have shown that quantitative methods based on the use of fluorogenic peptides cannot be directly applied to plant homogenates, due to the presence of interfering proteases with cleavage specificities similar to that of the proteasome. To overcome this, we developed a simple and rapid fractionation procedure that efficiently separates most of the non-proteasomal chymotryptic enzymes, such that proteasome activity can be easily measured. We go on to demonstrate that in vivo treatment of tobacco protoplasts with high concentrations of three potent proteasome inhibitors can only partially suppress proteasomal chymotrypsin-like activity, resulting in the incomplete stabilisation of the protein toxin ricin A chain (RTA), a known endoplasmic reticulum-associated degradation (ERAD) substrate that normally undergoes extensive cytosolic degradation. We therefore conclude that negative results obtained using proteasome inhibitors in tobacco protoplasts and possibly other types of plant cells should be interpreted with a degree of caution.

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Acknowledgements

The authors wish to thank Spencer Whitney (Australian National University, Canberra, Australia) for providing the antiserum against tobacco Rubisco, Anna Paola Casazza (Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milano, Italy) for providing the anti-LHCB1 antibodies, Herman S. Overkleeft (Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands) for providing the fluorescent probe MV151 and for helpful suggestions, Richard Vierstra and David Gemperline (University of Wisconsin, Madison, USA) for critical reading of the manuscript. This work was supported by a grant of the Ricerca Locale dell’Università degli Studi di Torino to P.C., by the Program FILAGRO - Strategie innovative e sostenibili per la filiera agroalimentare (Accordo quadro CNR-Regione Lombardia), and through a UK Biotechnology and Biological Sciences Research Council studentship awarded through L.M.R. for R.S.M.

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  1. Richard S. Marshall

    Present address: Department of Genetics, University of Wisconsin, 425 Henry Mall, Madison, WI, 53706, USA

Authors and Affiliations

  1. Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, Largo Paolo Braccini 2, 10095, Grugliasco, Torino, Italy

    Paolo Cascio, Fulvia Cerruti & Mary Raule

  2. Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133, Milano, Italy

    Richard S. Marshall, William Remelli & Aldo Ceriotti

  3. School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK

    Lynne M. Roberts

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  1. Paolo Cascio
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Correspondence to Paolo Cascio or Aldo Ceriotti.

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Cascio, P., Cerruti, F., Marshall, R.S. et al. A Quantitative Method to Monitor the Efficacy of Inhibitors Against the Chymotrypsin-Like Activity of the Proteasome in Tobacco Leaf Protoplasts. Plant Mol Biol Rep 33, 829–840 (2015). https://doi.org/10.1007/s11105-014-0793-2

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