Interaction of the RING finger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes

The U-box domain has been suggested to be a modified RING finger motif wher e the metal-coordinating cysteines and histidines have been replaced with o ther amino acids. Known U-box-containing proteins have been implicated in t he ubiquitin/proteasome system. In a search for proteins interacting with t he ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-b ox containing protein, termed UIP5, that is exclusively found in the nucleu s as part of a nuclear dot-like structure. Interaction between UbcM4 and UI P5 was observed in vivo and in vitro with bacterially expressed proteins. I n addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analys is showed that the U-box, like the RING finger in other proteins, forms the physical basis for the interaction with E2 enzymes. Further support for th e structural similarity between U-box and RING finger comes from the observ ation that, in both cases, the same regions within the UbcM4 molecule are r equired for interaction, Our results establish at the molecular level a lin k between the U-box and the ubiquitin conjugating system and strongly sugge st that proteins containing U-box domains are functionally closely related to RING finger proteins.