title: Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system.
abstract: Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D007768:Lactalbumin / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011817:Rabbits; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D006609:High Mobility Group Proteins; D012346:RNA, Transfer, Amino Acyl; D012709:Serum Albumin; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D012343:RNA, Transfer; D014360:Trypsin Inhibitor, Kunitz Soybean; D012259:Ribonuclease, Pancreatic; D008836:Micrococcal Nuclease; D009113:Muramidase