The ubiquitin-proteasome system (UPS) targets numerous cellular proteins for degradation. It is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic cellular processes. Degradation of a protein via the ubiquitin-proteasome pathway involves two discrete and successive steps: (1) tagging of the substrate by covalent attachment of multiple ubiquitin molecules to synthesize the polyubiquitin chain proteolytic signal and (2) degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin catalyzed by ubiquitin-recycling enzymes (DUBs). Conjugation of ubiquitin to the protein substrate proceeds via a three-step cascade mechanism. Initially, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction resulting in a high-energy thiol ester intermediate. Subsequently, this intermediate is transferred to a member of the ubiquitin-carrier proteins family of enzymes, E2 (also known as a ubiquitin-conjugating enzyme [UBC]). Finally, from E2, the activated ubiquitin moiety is attached to the substrate that is specifically bound to an E3, a member of the ubiquitin-protein ligase family of proteins. By successively adding additional activated ubiquitin moieties to internal Lys residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized. The degradation signal that is recognized by the 26S proteasome complex is made of a Lys48 polyubiquitin chain. In contrast, monoubiquitination or polyubiquitination with chains linked together via Lys63 serve as nonproteolytic signals in intracellular trafficking, DNA repair, activation of transcription and other signal transduction pathways.