MULTIUBIQUITIN CHAINS LINKED THROUGH LYSINE-48 ARE ABUNDANT IN-VIVO AND ARE COMPETENT INTERMEDIATES IN THE UBIQUITIN PROTEOLYTIC PATHWAY

A prerequisite for the selective degradation of intracellular proteins by the ubiquitin-dependent proteolytic pathway is the attachment of a chain of ubiquitin monomers to the targeted protein. In these multiub iquitin chains, the carboxyl-terminal glycine 76 of ubiquitin is linke d via an isopeptide bond to the epsilon-amino group of lysine 48 in th e adjacent ubiquitin. It remains to be determined whether these chains are preassembled and attached en masse to the target, are made by seq uential conjugation of ubiquitin monomers to ubiquitins already linked to the protein, or both. Using the 20-kDa ubiquitin-conjugating enzym e TaUBC7 from wheat, we have generated free, glycine 76-->lysine 48-li nked multiubiquitin chains (Ubq(n)), and have individually purified Ub q(n) species (n less-than-or-equal-to 6) by anion-exchange, high press ure liquid chromatography. The migration of these chains during SDS-po lyacrylamide gel electrophoresis was indistinguishable from those of m ajor ubiquitin immunoreactive proteins in cell lysates from a variety of eukaryotes suggesting that free, multiubiquitin chains are abundant in vivo. One of these chain members (Ubq2) was purified from wheat an d was demonstrated via amino acid sequence analysis of tryptic fragmen ts to consist of two ubiquitin monomers joined via a glycine 76-->lysi ne 48 linkage. We also show in vitro that purified Ubq2 and Ubq4 are c ompetent in activation by ubiquitin-activating enzyme (E1), in transfe r to E2s, and in ubiquitin-protein ligase (E3)-independent conjugation to other Ubq(n) species and to histones H2A/B. These data demonstrate that multiubiquitin chains exist as free, functional structures in vi vo and support the possibility that at least a portion of free ubiquit in is preassembled into multiubiquitin chains prior to its attachment to proteolytic substrates.