Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways.

In certain physiological or pathological states (e.g., starvation, heat shock, or muscle atrophy) and upon drug treatments, the overall rate of protein degradation in cells may increase or decrease. These adaptations and pathological responses can occur through alterations in substrate flux through the ubiquitin-proteasome pathway (UPP), the autophagy-lysosomal system, or both. Therefore, it is important to precisely measure the activities of these degradation pathways in degrading cell proteins under different physiological states or upon treatment with drugs. In particular, proteasome inhibitors have become very important agents for treating multiple myeloma and very useful tools in basic research. To evaluate rigorously their efficacy and the cellular responses to other inhibitors, it is essential to know the degree of inhibition of protein breakdown. Unfortunately, commonly used assays of the activities of the UPP or autophagy rely on qualitative, indirect approaches that do not directly reflect the actual rates of protein degradation by these pathways. In this chapter, we describe isotopic pulse-chase methods to directly measure overall rates of protein degradation in cells by radiolabeling cell proteins and following their subsequent degradation to radioactive amino acids, which diffuse from cells into the medium and can be easily quantitated. While pulse-chase methods have often been used to follow degradation of specific proteins, the methods described here allow quantification of the total cellular activity in degrading either long-lived proteins (the great bulk of cell constituents) or the fraction with short half-lives. Moreover, by use of specific inhibitors of proteasomes or lysosomes, it is also possible to measure precisely the total contributions of the UPP or lysosomal proteases. These approaches have already been proven very useful in defining the effects of inhibitors, growth factors, nutrients, ubiquitination, and different proteasome activators on overall proteolysis and on substrate flux through the proteasomal and lysosomal pathways.