Highlights d Respiratory dysfunction impairs 26S proteasome assembly and activity d Aspartate deficiency inhibits protein synthesis and 26S proteasome activity via mTORC1 d Aspartate restores 26S activity via upregulation of proteasome assembly factors d Addition of pyruvate overcomes resistance to bortezomib in respiration-deficient cells

Summary This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes. For complete details on the use and execution of this protocol, please refer to Meul et al. (2020) .

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