Classes/Examples of Ubiquitin Ligases (E3s). (I) HECT-Domain E3. E6-AP ubiquitin ligase in combination with E6 protein and one of the two E2s (UbcH5 or UbcH7) ligates ubiquitin to the p53 tumor suppressor protein. (IIA) Single-subunit RING Finger E3. Mdm2 ligates ubiquitin to PSD 95 with the help of an E2 enzyme. (IIBi) Multi-subunit RING finger E3. SCF ligases contain the substrate recognition site on an F-box protein. Skp1 is an adaptor that joins the F box protein to Cul1. Ring finger domain is on Rbx 1. The E2 is Ubc3. Cul 1 is modified by Nedd 8, a ubiquitin- like protein leading to an increase in the activity of the ligase complex. The substrate is phosphorylated (diamonds) IκBα. (IIBii) Multi-subunit RING finger E3. APC is a more complex example of multi-subunit RING finger E3s and has a subunit composition distinct from that of SCF. Cdc20 protein in APC has the substrate (Cyclin) recognition site. The RING finger domain is on APC11. The E2s Ubc 11 or UbcX can function with the APC ligase. In addition, several adaptor proteins, some labeled (Cdc27, Cdc23, APC 1, Cdc16) and some unlabeled, interact with Cdc20 and APC11. Diamonds on the adaptor subunits indicate phosphorylation. Polyubiquitin chain is shown on the substrates in each panel. In all panels, E2s are light blue, RING finger domains are dark blue and the substrates are purple in color.

The ubiquitin-proteasome pathway. In this proteolytic pathway, ubiquitin (single ubiquitin molecule is represented by open circles with straight tails) is selectively and covalently liked to the substrate. The enzymatic process of attaching ubiquitin to substrates is called ubiquitination or ubiquitin conjugation and depends on the action of three different classes of enzymes E1, E2 and E3. First ubiquitin is activated by E1 to form a ubiquitin-AMP intermediate. Activated ubiquitin (light orange color-filled circles with straight tails) is passed on to an E2 (ubiquitin carrier enzyme). Some E3s (ubiquitin ligases) such as HECT ligases accept ubiquitin as a thiol intermediate and then ligate it to the substrate. The RING ligases bring the E2 and substrate together and facilitate the transfer of ubiquitin from the E2 to the substrate. To the ubiquitin attached to substrate another ubiquitin is attached and thus through successive linkages of ubiquitin a polyubiquitin chain forms (conjugated ubiquitin is represented by dark orange color-filled circles and a branched pattern of tails). Polyubiquitinated substrates are degraded by a proteolytic complex called the 26S proteasome in an ATP-dependent reaction. Ubiquitin is not degraded but the polyubiquitin chain is disassembled and ubiquitin is recycled by deubiquitinating enzymes (DUBs). Before being committed to be degraded by the proteasome, ubiquitination is reversible. DUBs can disassemble the polyubiquitin chain if a substrate is ubiquitinated erroneously and prevent the degradation of the substrate.