Abstract
The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent protein-Tom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Blotting, Western
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism
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Cell Line
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Chromatography, Gel
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Clathrin / chemistry
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Clathrin / metabolism
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Cytoplasm / metabolism
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DNA, Complementary / metabolism
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / metabolism
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Gene Library
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins
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Humans
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Interleukin-1 / metabolism
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Intracellular Signaling Peptides and Proteins*
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Luminescent Proteins / metabolism
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Mice
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Microscopy, Fluorescence
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Molecular Sequence Data
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Mutation
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NIH 3T3 Cells
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Plasmids / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Proteins / chemistry
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Proteins / physiology*
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Signal Transduction
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Transfection
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Two-Hybrid System Techniques
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Ubiquitin / chemistry
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Ubiquitin / metabolism
Substances
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Carrier Proteins
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Clathrin
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DNA, Complementary
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Interleukin-1
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Intracellular Signaling Peptides and Proteins
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Luminescent Proteins
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Proteins
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Recombinant Proteins
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TOLLIP protein, human
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TOM1 protein, human
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TOM1 protein, mouse
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Tollip protein, mouse
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Ubiquitin
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Green Fluorescent Proteins
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Glutathione Transferase