Modification of proteins by the covalent attachment of ubiquitin plays a fundamental role in the control of many biological processes including cell cycle regulation, transcription, DNA repair and apoptosis. Substrate proteins are often modified by the attachment of polyubiquitin chains consisting of several ubiquitin moieties linked to each other via isopeptide bond formation. Ubiquitin contains 7 lysine residues and each of these lysines can be used for polyubiquitin chain formation. Importantly, it appears that the actuel lysine residue of ubiquitin used for ubiquitin-ubiquitin conjugation determines the biochemical/biological function of the respective polyubiquitin chain. For example, K48-linked polyubiquitin chains serve as a signal to target modified proteins for degradation by the proteasome, while K63-linked polyubiquitin chains have been linked to non-proteolytic processes. An attractive possibility is that the different function(s) of different polyubiquitin chains are mediated by proteins that selectively interact with the respective chain. However, with the exception of proteins interacting with K48-linked polyubiquitin chains, little is known about the identity of such proteins. In this project, a series of chemically defined polyubiquitin chains that differ by the lysine residue used for ubiquitin-ubiquitin conjugation will be synthesized by chemical and biochemical means. The different chains will be used to generate antibodies and to isolate and characterize proteins that selectively recognize distinct polyubiquitin chains in a linkage-specific manner. Thus, the studies will contribute to the elucidatioin of the biochemical and physiological function(s) of different polyubiquitin chains.