The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.
Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-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 AlixDeltaC (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 AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC 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.
Katoh
Keiichi
K
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shibata
Hideki
H
Suzuki
Hidenori
H
Nara
Atsuki
A
Ishidoh
Kazumi
K
Kominami
Eiki
E
Yoshimori
Tamotsu
T
Maki
Masatoshi
M
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D018797:Cell Cycle Proteins; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins
C478397:CHMP4B protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; C117020:PDCD6IP protein, human; C492144:Pdcd6ip protein, mouse; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C083890:SNF7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human