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.
Highlights
The endosome system is involved in the trafficking and sorting of plasma membrane proteins that have undergone endocytosis and newly synthesized proteins for which the final destination is the lysosome [1, 2]
Proteins in the second, including Hrs and Vps27, each contain a VHS domain, an FYVE (Fab1b, YOTB, Vac1p, and EEA1) domain that can bind to membrane lipid, a coiled-coil domain that can bind with STAM, a ubiquitin-interacting motif (UIM), and a clathrin-binding motif that is recognized by clathrin [8, 9]
We found that Tom1 directly associates with Tollip to form a complex and that Tom1, as well as Tollip, is capable of directly binding with polyubiquitin chains
Summary
The endosome system is involved in the trafficking and sorting of plasma membrane proteins that have undergone endocytosis and newly synthesized proteins for which the final destination is the lysosome [1, 2]. Tom1 Binds with Tollip to Form a Complex inside the Cell—To obtain definitive evidence of interaction between Tom1 and Tollip, we co-expressed Flag-tagged Tom1 and T7tagged Tollip in HEK293T cells, and the extracts of transfected cells were subjected to immunoprecipitation using anti-Flag tag antibody and to Western blotting with anti-T7 tag antibody to detect interaction (Fig. 1A).
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