Ubiquitin, a protein of Mr = 8,500, is encountered in all living cells, yet its function is poorly understood. Some of the reactions of cellular proteins with ubiquitin, requiring ATP as the direct energy source, lead to proteolytic cleavage, while the others result in the formation of covalent ubiquitin-protein conjugates. Ubiquitin-linked proteolysis is dependent on the age of the cell, but the mechanism of the process is unknown. It is of primary importance in understanding the biochemistry of protein degradation to determine what structural features of proteins are responsible for """"""""ubiquination"""""""" and the fate of the conjugate. Model proteins of well-known structure will be used as the substrates for """"""""ubiquination"""""""". The conjugate formation and proteolytic cleavage will be separated in time by consecutive addition of appropriate enzymes to the incubation mixture. Ubiquitin-linked proteolysis is differently expressed in various cells and organs. By using differentially radiolabeled ubiquitin-protein conjugates as substrates, the contribution of ubiquitin-dependent proteolysis will be examined in liver and fibroblasts (normal, aged, and virus-transformed) cells as a function of cell age and type. The existence of stable conjugates between ubiquitin and specific proteins raises questions concerning the specificity of the conjugation and degradation. The proteins from rat liver or fibroblast homogenates, in which pools of rapidly and slowly degraded proteins are differentially radiolabeled, will be resolved by a two-dimensional gel electrophoresis. To find out if one of these classes of proteins is more prone to accumulation of stable conjugates, the specific conjugates will be detected with radiolabeled antibody against ubiquitin using immunoblotting techniques. In vivo studies on subcellular localization of ubiquitin will be undertaken to learn if it varies with the cell age or nutritional state. The binding of micro-injected, fluorescently labeled antibody against ubiquitin will be detected in fibroblast using digital imaging fluorescence microscopy. The experimental goals will advance the understanding of the specificity, mechanism, and, possibly, some of the functions of the complex biochemical systems utilizing ubiquitin.
