Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system

Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Ubiquitin-marking appears to be important to the progression of cells from one stage to another of the cell cycle; it may also be involved in gene activation. Here we show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of 125I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNAHis, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.