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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 AlixΔC (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 …