Conjugation of ubiquitin to denatured hemoglobin is proportional to the rate of hemoglobin degradation in HeLa cells.

Ubiquitin was radioiodinated and introduced into HeLa cells by the erythrocyte-mediated fusion procedure. Fractionation of injected HeLa cells and subsequent NaDodSO4/polyacrylamide gel electrophoresis showed that HeLa nuclei contained two major labeled proteins: ubiquitin and the histone H2A-ubiquitin conjugate, protein A24. HeLa cytosol contained ubiquitin and a series of ubiquitin-protein conjugates of diverse molecular weights. When injected HeLa cells were treated with phenylhydrazine to denature the cotransferred hemoglobin, a series of prominent ubiquitin-globin conjugates appeared. The identity of these conjugates was established by microinjection experiments in which both proteins were labeled. At low doses of phenylhydrazine, the intracellular concentration of globin-ubiquitin conjugates was proportional to the rate of hemoglobin degradation. This result, together with the observation that ubiquitin conjugation to globin is markedly enhanced by phenylhydrazine-induced denaturation of hemoglobin, provides support for the hypothesis that the covalent attachment of ubiquitin to proteins signals proteolysis.