The role of ubiquitin in ATP-dependent proteolysis.

Radiolabeled ubiquitin was introduced into HeLa cells by red cell-mediated fusion procedure. The nuclei contained two major labeled proteins, ubiquitin and protein A24. The cytosol contained ubiquitin and a series of ubiquitin-protein conjugates. When the cotransferred hemoglobin was denatured by phenylhydrazine, a series of prominent ubiquitin-globin conjugates appeared. The concentration of globin-ubiquitin conjugates was proportional to the rate of hemoglobin degradation. These results provide support for the hypothesis that the covalent attachment of ubiquitin to proteins signals proteolysis. However, direct ubiquitin attachment to substrates does not appear to be mandatory for proteolysis. Guanidinated (G) proteins, which have blocked lysine residues, were used as potential substrates for ATP-dependent proteolysis in rabbit reticulocyte lysates. G-lysozyme was degraded by an ATP-dependent process, yet ubiquitin conjugation to this protein was not observed. In contrast, conjugates between ubiquitin and lysozyme were readily detected. A ubiquitin-free fraction of the lysate was obtained which degraded both G-lysozyme and lysozyme by an ATP-dependent process that was stimulated by ubiquitin. Neither ubiquitin nor ATP alone significantly stimulated the formation of labeled adducts in ubiquitin-free lysates incubated with the labeled proteins. When both ubiquitin and ATP were added to the reaction mixtures, G-lysozyme-ubiquitin conjugates were not observed; whereas, lysozyme-ubiquitin conjugates were readily detected. These observations cast doubt on the hypothesis that ubiquitin has a unitary role as a covalent marker in ATP-dependent proteolysis.