Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7

The beet domain protein family was originally identified by sequence similarity of its members to the C-terminal region of E6-AP, an E3 ubiquitin-protein ligase. Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin, a necessary intermediate step in E6-AP-dependent ubiquitination, it was proposed that members of the beet domain family in general have E3 activity. The beet domain is approximately 350 amino acids in length, and we show here that the beet domain of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation. Furthermore, the human genome encodes at least 20 different beet domain proteins, and in further support of the hypothesis that beet domain proteins represent a family of E3s, several of these are shown to form thioester complexes with ubiquitin. In addition, some beet domain proteins interact preferentially with UbcH5, whereas others interact with UbcH7, indicating that human beet domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic pathways.