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.
D000818:Animals; D002417:Cattle; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D009698:Nucleoproteins / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D013950:Thymus Gland; D014452:Ubiquitins
D002868:Chromosomal Proteins, Non-Histone; D006454:Hemoglobins; D007457:Iodine Radioisotopes; D009698:Nucleoproteins; D014452:Ubiquitins