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J. Biol. Chem., Vol. 278, Issue 52, 52865-52872, December 26, 2003

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Tom1, a VHS Domain-containing Protein, Interacts with Tollip, Ubiquitin, and Clathrin^*

Megumi Yamakami, Tamotsu Yoshimori, and Hideyoshi Yokosawa¶

From the Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 and the Department of Cell Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka-ken 411-8540, Japan

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.

Received for publication, June 25, 2003 , and in revised form, September 29, 2003.

^* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-11-706-3754; Fax: 81-11-706-4900; E-mail: yoko{at}pharm.hokudai.ac.jp.

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