The Ubiquitin-Proteasome System

Intracellular protein degradation has been studied for more than half a century, and it became clear early on that such degradation is highly selective, with individual protein half-lives ranging from minutes to years. Moreover, much of this degradation was found to be energy-dependent, despite the exergonic nature of peptide-bond cleavage. This energy dependence derives from the dual requirements of high substrate specificity and substrate-protein unfolding to make the polypeptide backbone fully accessible for proteolytic cleavage. Most regulated protein degradation in eukaryotes is executed by the ubiquitin–proteasome system. Polyubiquitylation of substrates by specific enzymes provides the major source of selectivity in the system (Figure 1), whereas the 26S proteasome complex performs the protein unfolding that is necessary for processive cleavage of the tagged proteins into short peptides (Figure 2). In addition, ubiquitin ligation can function independently of the proteasome by directing certain proteins (usually membrane proteins) to the lysosome or vacuole for proteolysis. Conversely, proteasomes can degrade some proteins without their prior modification by ubiquitin

Figure 1. Substrate proteins, destined for elimination, are initially attached to polymers of the highly conserved ubiquitin (Ub) protein. This covalent modification of the substrate targets the conjugated protein to a multicatalytic protease complex, the 26S proteasome. The Ub attachment site in substrate proteins is commonly a Lys side chain. A well-defined series of enzymes orchestrates the attachment of mono- and polyubiquitin to proteins (see figure). Ub is first activated in an ATP-consuming reaction by an E1 Ub-activating enzyme, to which it becomes attached by a high-energy thioester bond. Subsequently, the activated Ub is transferred to the active site Cys of a second protein, an E2 ubiquitin-conjugating enzyme. With the aid of a third enzyme, called E3 or ubiquitin-protein ligase, E2 catalyses the transfer of (poly)ubiquitin onto the protein that is destined for degradation.

E3 is the most important enzyme in determining the specificity of substrate ubiquitylation. There are two major classes of mechanistically distinct E3 enzymes, characterized by the RING (or RING-like) and HECT domains. Both types of E3 enzymes are alike in their ability to establish selective substrate binding. The RING finger uses Cys and His residues to coordinate a pair of zinc ions in a characteristic arrangement (not shown). A smaller set of E3 enzymes contain a domain called the U box, which is a degenerate version of the RING-finger that achieves the same general fold without coordinating any metal ions. RING and RING-like E3 enzymes bind to both the E2 enzyme and the substrate, and catalyse the transfer of Ub directly from the E2 enzyme to the substrate. Unlike RING and U-box E3 enzymes, the HECT E3 enzymes have a more direct catalytic role in substrate ubiquitylation. The activated Ub of the Ub–E2 enzyme thioester is transferred to a conserved Cys residue in the HECT domain of the E3 before finally being transferred to a substrate.

Ubiquitylation is reversed by de-ubiquitylating enzymes (DUBs) that remove ubiquitin from proteins and disassemble polyubiquitin chains. DUBs provide additional regulatory control before protein degradation, and they are also fundamental for maintaining a sufficient pool of free ubiquitin molecules in the cell.

Figure 2. Once modified by a polyubiquitin chain of at least four ubiquitins (Ub), the substrate protein is able to bind either directly to intrinsic Ub receptors in the 19S regulatory complex of the 26S proteasome (A) or to adaptor proteins that bear both polyubiquitin-binding and proteasome-binding domains (B). Exactly why certain polyubiquitin-modified substrates must be shuttled to the proteasome by adaptor proteins and others can associate directly with polyubiquitin-binding subunits in the regulatory complex of the proteasome is not fully understood. Binding of the substrate protein to the proteasome is followed by protein unfolding by the half-dozen ATPases that encircle the pore of the proteasome catalytic core, removal of the polyubiquitin chain by proteasome-associated deubiquitylating enzymes (DUBs), and translocation of the unfolded protein into the central proteolytic chamber, where it is cleaved into short peptides (C).


Ravid T, Hochstrasser MDiversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol. 2008;9(9):679-90.