The 1, 2, 3's of protein ubiquitination. Simplified schematic shows the three enzymatic activities associated with the central paradigm of protein ubiquitination: E1, the Ub-activating enzyme; E2, the Ub-conjugating enzyme; and E3, the Ub ligase. Mechanistically, there are two types of E3s, the RING/UBox-type E3s that effect transfer of Ub directly from the active site of an E2 to a lysine residue of a substrate, and the HECT-type E3s that facilitate Ub transfer from an E2 to substrate via a thioester intermediate on the E3. Auto-ubiquitination of the E3 is also observed in some cases and can be used as a proxy signal to assay the activity of an E2/E3 pair in the absence of a substrate. The best studied fate of poly-ubiquitinated substrates is degradation by the proteasome (not depicted).