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. com cyclins, which control the transitions through various steps in the cell cycle2, 3. Moreover, a protein’s rate of degradation may vary dramatically under different physiological conditions. Proteolysis thus serves as a timing device that, by degrading key proteins, alters rates of gene transcription, fluxes through biochemical pathways, and other cell processes2–4. Another essential function of intracellular proteolysis is to serve as a quality control mechanism that selectively destroys proteins with abnormal conformations, as may arise by mutations, failure to fold correctly, or postsynthetic damage. In fact, many biochemists, molecular biologists, and even investors in biotechnology were first introduced to intracellular proteolysis when attempts to produce foreign proteins in Escherichia coli proved unsuccessful because the bacteria rapidly hydrolyzed the abnormal polypeptide. The successful production of many recombinant proteins required the development of protease-deficient organisms and extremely efficient expression systems that overwhelm the bacteria’s proteolytic apparatus. It is obvious that such selective, regulated proteolysis would be impossible if the cytosol contained the simple types of proteases that function in the extracellular environment. Within cells, such enzymes would be extremely damaging and would quickly destroy essential cell constituents. Instead, proteolysis in eukaryotic cells relies on enzyme systems that specifically mark substrates for degradation by the very large proteolytic complex, the 26S proteasome, in which proteolysis is isolated within an internal compartment, the 20S proteasome, to which access is tightly regulated5. Specificity in this pathway is achieved largely by a group of enzymes that covalently modify proteins by linkage to the small polypeptide ubiquitin (see Fig. 1) to target them to the 26S proteasome.

The first step in this pathway is the ATP-dependent activation of the carboxyl end of ubiquitin by the enzyme, E1. The resulting highly reactive ubiquitin thiolester is then transferred to one of the cell’s many ubiquitincarrier proteins (E2s), which in turn transfers the ubiquitin to a lysine residue on the substrates. This step requires an E3, or ubiquitin protein ligase, which binds the polypeptide substrate and catalyzes the attachment of a chain of ubiquitin molecules. Cells may contain over a hundred distinct E3s, which, in concert with a specific E2, preferentially ubiquinate different groups of proteins. In the present paper, Dantuma et al. create short-lived fusion proteins containing the green fluorescent protein (GFP) marker for the purpose of assessing rates of protein breakdown. By correlating the level of fluorescence with proteolytic breakdown, they are able to assay proteasome-dependent degradation of the fusion proteins in mam-