a| Type 1 (basic) and type 2 (hydrophobic) destabilizing residues of the N-end rule pathway are shown in a single letter code. b| Cleavage by endopeptidases can lead to the positioning of a destabilizing residue (X) at the N terminus of the truncated protein. c| Cleavage between Met and Cys by methionine aminopeptidase (indicated by a lightning bolt) can lead to protein destabilization when followed by addition of Arg, a type 1 destabilizing residue (see panel d). d| Modification of Asn to Asp (or Gln to Glu) by specific deamidases can lead to the addition of Arg by arginyltransferase (upper panel). Oxidation of the N-terminal Cys residue (marked by Asterisk) can similarly lead to protein arginylation and degradation (lower panel).

a| Overall architecture of the Skp1-Fbw7-CycE C-terminal phosphodegron (CycE^degC) complex, with the secondary structure elements of Skp1, F-box, and linker domains labelled. b| CycE^degC binds across the narrow face of the Fbw7 β-propeller structure. The two phosphorylated residues Trp380 and Ser384 are shown. The eight Fbw7 blades and the strands for one blade are labelled by numbers and letters, respectively.

a| Helical-wheel representation of a region (residues 18-36) of the yeast α2 Deg1 degron that is predicted to form part of a coiled-coil structure. Residues whose mutation inhibited Deg1-mediated proteolysis are marked in bold. b| A model for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) organization in the endoplasmic reticulum (ER) membrane. The sterol-sensing domain is marked in orange. c| 3D structure of the Man3GlcNAc2 oligosaccharide attached to Asn34 of RNase B. The β-1,4-linked dimer of glucosamine (chitobiose), which functions as a binding site for SCF^Fbs1, is marked in red.

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

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 (see ) or to adaptor proteins that bear both polyubiquitin-binding and proteasome-binding domains (see ) . 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 (see ).