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J. Biol. Chem., Vol. 278, Issue 40, 39104-39113, October 3, 2003

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The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting^*

Keiichi Katoh , Hideki Shibata , Hidenori Suzuki , Atsuki Nara , Kazumi Ishidoh ¶, Eiki Kominami ¶, Tamotsu Yoshimori  and Masatoshi Maki  ||

From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, the Department of Cell Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan and the ^¶Department of Biochemistry, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca²⁺-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixC were co-localized. SKD1^E235Q, a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1^E235Q as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1^E235Q in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.

Received for publication, February 14, 2003 , and in revised form, July 14, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB100261 and AB100262 (at the early stage of this study, we designated CHMP4a and CHMP4b as Shax2 and Shax1, respectively).

^* This work was supported by a Grant-in-Aid for Scientific Research B (to M. M.) and a Grant-in-Aid for Young Scientists B (to H. S.). 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.: 81-52-789-4088; Fax: 81-52-789-5542; E-mail: mmaki{at}agr.nagoya-u.ac.jp.

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