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

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.