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 where the metal-coordinating cysteines and histidines have been replaced with other amino acids. Known U-box-containing proteins have been implicated in the ubiquitin/proteasome system. In a search for proteins interacting with the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-box containing protein, termed UIP5, that is exclusively found in the nucleus as part of a nuclear dot-like structure. Interaction between UbcM4 and UIP5 was observed in vivo and in vitro with bacterially expressed proteins. In addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analysis 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 the structural similarity between U-box and RING finger comes from the observation that, in both cases, the same regions within the UbcM4 molecule are required for interaction. Our results establish at the molecular level a link between the U-box and the ubiquitin conjugating system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany.
E
E
Pringa
G
G
Martinez-Noel
U
U
Muller
K
K
Harbers
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008025:Ligases / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D034961:Antigens, Nuclear; D001324:Autoantigens; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C431153:UBOX5 protein, human; D014452:Ubiquitins; C066642:SP100 protein, human; D006639:Histidine; D012313:RNA; C117188:Ube2l3 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D003545:Cysteine