Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Antigens, Nuclear*
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Autoantigens / metabolism
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Blotting, Northern
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism*
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Cell Line
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Cell Nucleus / metabolism*
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Cysteine / chemistry
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Cysteine Endopeptidases / chemistry*
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Cysteine Endopeptidases / metabolism
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HeLa Cells
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Histidine / chemistry
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Humans
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Immunohistochemistry
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Ligases / chemistry
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Ligases / metabolism
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Mice
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Microscopy, Fluorescence
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Molecular Sequence Data
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Multienzyme Complexes / chemistry*
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Multienzyme Complexes / metabolism
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Nuclear Proteins / chemistry*
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Nuclear Proteins / metabolism*
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Plasmids / metabolism
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Proteasome Endopeptidase Complex
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Protein Binding
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Protein Structure, Tertiary
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RNA / metabolism
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Tissue Distribution
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Transfection
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Two-Hybrid System Techniques
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Ubiquitin-Conjugating Enzymes*
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Ubiquitins / chemistry*
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Ubiquitins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Antigens, Nuclear
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Autoantigens
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Carrier Proteins
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Multienzyme Complexes
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Nuclear Proteins
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Recombinant Proteins
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UBOX5 protein, human
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Ubiquitins
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SP100 protein, human
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Histidine
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RNA
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Ube2l3 protein, mouse
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Ubiquitin-Conjugating Enzymes
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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Ligases
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Cysteine