The ubiquitin (Ub)-mediated protein degradation pathway involves three sequential enzymatic steps that facilitate the conjugation of Ub to specific protein substrates. The first step requires ATP-dependent activation of the C-terminus of Ub and the assembly of multi-Ubs by Ub-activating enzyme E1. The ubiquitin-conjugating enzyme E2, catalytic (UBCc) domain, is then conjugated to Ubs, through a thiol-ester linkage between a conserved cysteine and the C-terminus of Ub, to generate an intermediate Ub-E2 complex. Then the E3, a ligase, catalyzes the transfer of Ub from E2 to the appropriate substrate. This pathway regulates many fundamental cellular processes. There are also other E2s which form thiol-ester linkages without the use of E3s as well as several UBC homologs (TSG101, Mms2, Croc-1 and similar proteins), which lack the active site cysteine essential for ubiquitination and appear to function in DNA repair pathways.