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Title of Journal: Plant Mol Biol Rep

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Abbravation: Plant Molecular Biology Reporter

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Springer US

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DOI

10.1007/s00520-009-0741-0

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1572-9818

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A Quantitative Method to Monitor the Efficacy of I

Authors: Paolo Cascio Fulvia Cerruti Richard S Marshall Mary Raule William Remelli Lynne M Roberts Aldo Ceriotti
Publish Date: 2014/09/18
Volume: 33, Issue: 4, Pages: 829-840
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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 chymotrypsinlike 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 nonproteasomal 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 chymotrypsinlike activity resulting in the incomplete stabilisation of the protein toxin ricin A chain RTA a known endoplasmic reticulumassociated 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 cautionThe 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 antiLHCB1 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 PC by the Program FILAGRO Strategie innovative e sostenibili per la filiera agroalimentare Accordo quadro CNRRegione Lombardia and through a UK Biotechnology and Biological Sciences Research Council studentship awarded through LMR for RSM

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  2. An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides
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  5. Genome-wide Comparisons of Gene Expression for Yield Heterosis in Maize
  6. Transcriptional Regulation of the Rice Phytase Gene OsPHY1 by Several Phytohormones and Osmotic Stresses Using Promoter-GUS Analysis
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