Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells.

The lysosome and 26S proteasome represent the two major proteolytic machines in eukaryotic cells (1). While the lysosome deals mainly with nonselective proteolysis, the 26S proteasome handles the majority of regulated proteolysis. The 26S proteasome is a multisubunit protease that degrades the substrate into small peptides in an ATPdependent manner (2,3). The proteasome has three peptidase activities, including (i) chymotrypsin-like, (ii) trypsin-like, and (iii) peptidylglutamylpeptide hydrolase activities (2). To demonstrate that a protein is a substrate of the proteasome in vivo, the stability of the protein is often examined and compared in the presence or absence of proteasome inhibitors (e.g., MG132), short peptide aldehydes that block active sites of the proteasome (4). Use of the budding yeast Saccharomyces cerevisiae as a model system has been instrumental in uncovering mechanistic attributes and the physiologic functions of the proteasome. However, the use of proteasome inhibitors in wild-type S. cerevisiae cells is hampered by the impermeability of the cell wall or membrane (5). Therefore, mutant yeast strains (e.g., erg6Δ, pdr5Δ) with increased drug permeability or reduced drug efflux are required for experiments using proteasome inhibitors (5,6). A caveat to this approach is that mutation in ERG6 or PDR5 may directly or indirectly affect some cellular processes (e.g., increased import of sodium) (7,8) and protein stability. Furthermore, in some cases, the ERG6 or PDR5 genes must be deleted in another mutant background, a technically cumbersome step, to establish the involvement of the 26S proteasome in a particular process (e.g., transcription or telomere maintenance) (9,10). Recently, a method was developed involving brefeldin A, an antifungal agent often used to study protein trafficking from Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells