The U box is a modified RING finger — a common domain in ubiquitination

Eukaryotes use small polypeptides of the ubiquitin [1,2] and Apg12 [3] families as tags to target proteins for degradation by the proteasome. Ubiquitin ligation is a multi-step process that involves at least three classes of enzymes [1]. The E1 enzymes first charge ubiquitin in an ATP-dependent manner to form an E1–ubiquitin thioester intermediate. This activated ubiquitin is transferred to the active cysteine of an E2 enzyme and finally to an E3 enzyme. The E3 enzyme interacts directly with the substrate and mediates ubiquitin transfer to the amino group of a lysine. Recently, a new class of ubiquitination enzymes, E4, the prototype of which is the yeast protein UFD2, has been identified. E4 is required for the multi-ubiquitination of ubiquitin-conjugated proteins that makes them preferred substrates for degradation [4]. The UFD2 protein and its homologs in other eukaryotes share a conserved domain designated the ‘U box’. The U box mediates the interaction of UFD2 with ubiquitin conjugated proteins and therefore seems to be an essential functional unit of the E4 enzymes [4]. Here, we show by means of sequence-profile analysis that the U box is a derived version of the RING-finger domain that lacks the hallmark metal-chelating residues of the latter [5,6] but is likely to function similarly to the RING-finger in mediating ubiquitin-conjugation of protein substrates. A PSI-BLAST search [7] initiated with the U box of UFD2 recovered, in addition to other U-box proteins, several bona fide RING fingers, such as TRAF-6, RAG1 and COP1, within three iterations, with statistically significant expectation (E) values in the range 10–3–10–5. Surprisingly, however, most of the signature cysteines of the RING finger are not conserved in the U box. A reverse search with a position-specific weight matrix for the RING fingers against the complete protein sets of Saccharomyces cerevisiae and Caenorhabditis elegans and partial protein sets of Schizosaccharomyces pombe and Arabidopsis thaliana R132 Current Biology Vol 10 No 4