The long term goal of this research is to define the specificity, mechanism and physiological significance of the ubiquitin-dependent protein degradation system of eucaryotic cells. This soluble multi-enzyme system has been best characterized from reticulocytes where it appears to play a role in the degradation of abnormal proteins and intracellular organelles during maturation. The objective of this proposal is to provide a basic biochemical description of the protease(s) of the system and the metabolism of conjugated proteins by these proteases. Ubiquitin is conjugated to a large number of proteins in the cell by the formation of an isoamide bond between the carboxyl-terminus of ubiquitin and the amino groups of the target proteins. It is these conjugates which are thought to be substrates for the proteases of the system. Understanding of the proteolytic mechanisms has been hampered by the lack of pure, defined substrates. This has slowed the development of assays and, therefore, the purification of the proteases. The proposed work will address this problem by synthesizing and characterizing a number of these conjugates using the carboxyl-terminal acyl azide of ubiquitin. These substrates will be used to purify and characterize the components of the proteases, as well as to determine the specificity and architecture of the multi-enzyme complex which catalyzes these reactions. Additionally, the nature of the ATP requirement for proteolytic action will be examined. The knowledge gained from these studies should form the basis for the design of specific inhibitors of this system, as well as for the cloning and genetic manipulation of the component peptides of the system.
