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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

The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 40, Issue of October 3, pp. 39104 –39113, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting* Received for publication, February 14, 2003, and in revised form, July 14, 2003 Published, JBC Papers in Press, July 14, 2003, DOI 10.1074/jbc.M301604200 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; also named AIP1) was Alix (ALG-2-interacting protein X) is a 95-kDa protein 2 2 that interacts with an EF-hand type Ca -binding pro- found as a protein that interacts with the Ca -binding protein tein, ALG-2 (apoptosis-linked gene 2), through its C-ter- ALG-2 (apoptosis-linked gene 2) (1) by the yeast two-hybrid minal proline-rich region. In this study, we searched for method using mouse cDNA libraries by two independent proteins that interact with human AlixC (a truncated groups (2, 3). The interaction between Alix and ALG-2 was form not containing the C-terminal region) by using a shown to be Ca -dependent. Human Alix is a 95-kDa protein yeast two-hybrid screen, and we identified two similar that consists of 868 amino acids. It possesses no obvious struc- human proteins, CHMP4a and CHMP4b (chromatin- tural motifs except for coiled-coil regions and a long proline- modifying protein; charged multivesicular body pro- rich region on the C-terminal side. ALG-2 is one of the penta- tein), as novel binding partners of Alix. The interaction EF-hand Ca -binding proteins (4, 5) and interacts with the of Alix with CHMP4b was confirmed by a glutathione N-terminal domains of annexins VII and XI, which show some S-transferase pull-down assay and by co-immunopre- similarities with the proline-rich region of Alix (6, 7). ALG-2 cipitation experiments. Fluorescence microscopic anal- forms a homodimer as well as a heterodimer with another ysis revealed that CHMP4b transiently expressed in penta-EF-hand protein named peflin (8, 9). In a subcellular HeLa cells mainly exhibited a punctate distribution in fractionation experiment, most of Alix was recovered in the the perinuclear area and co-localized with co-expressed cytosolic fraction of fibroblast cells (3). Results of immunofluo- Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker pro- rescent microscopic analysis showed that overexpressed Alix teins, EEA1 (early endosome antigen 1) and Lamp-1 (ly- was present in the cytoplasm but was concentrated in the sosomal membrane protein-1), respectively. Transient perinuclear region and also localized at the cell periphery in overexpression of CHMP4b induced the accumulation of lamellipodia and filopodia (10). Moreover, proteomic analyses ubiquitinated proteins as punctate patterns that were have revealed that Alix is also present in extracellular mi- partly overlapped with the distribution of CHMP4b and crovesicles called exosomes, which are derived from dendritic inhibited the disappearance of endocytosed epidermal cells (11), and phagosomes isolated from macrophage-like growth factor. In contrast, stably expressed CHMP4b in cells (12). HEK293 cells was observed diffusely in the cytoplasm. Homologues of Alix have been found in many organisms. The Transient overexpression of AlixC in stably CHMP4b- Xenopus homologue of Alix, Xp95, was identified as a protein of expressing cells, however, induced formation of vesicle- 95 kDa that was phosphorylated from the first to second mei- like structures in which CHMP4b and AlixC were co- E235Q otic divisions during progesterone-induced oocyte maturation localized. SKD1 , a dominant negative form of the (13). Overexpression of Hp95, a human homologue of Xp95 AAA type ATPase SKD1 that plays critical roles in the identical to human Alix, induced G arrest, promoted detach- endocytic pathway, was co-immunoprecipitated with 1 ment-induced apoptosis (anoikis), and reduced tumorigenicity CHMP4b. Furthermore, CHMP4b co-localized with E235Q SKD1 as punctate patterns in the perinuclear area, in HeLa cells (14, 15). PalA, an Aspergillus nidulans Alix and Alix was induced to exhibit dot-like distributions homologue, is required for alkaline adaptation of the filamen- E235Q overlapped with SKD1 in HeLa cells. These results tous fungi whose pH regulation of gene expression is mediated suggest that CHMP4b and Alix participate in formation by the zinc finger transcription factor PacC (16, 17). Budding of multivesicular bodies by cooperating with SKD1. yeast has two Alix homologues, Rim20p (18) and Bro1 (19). Genetic analysis of Rim20p has revealed that Rim20p is essen- tial for processing of the zinc finger protein Rim101p, a yeast * This work was supported by a Grant-in-Aid for Scientific Research homologue of PacC (18). Bro1 mutations result in a tempera- 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 ture-sensitive osmoremedial growth defect (19). Although their of page charges. This article must therefore be hereby marked “adver- functions are not known, Alix homologues have also been found tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate in Caenorhabditis elegans (named YNK1 (20)) and in Arabi- this fact. TM The nucleotide sequence(s) reported in this paper has been submitted dopsis (GenBank accession number AC007591, protein TM to the GenBank /EBI Data Bank with accession number(s) AB100261 ID AAD3964201). and AB100262 (at the early stage of this study, we designated CHMP4a In addition to ALG-2, some binding partners of Alix have and CHMP4b as Shax2 and Shax1, respectively). been identified. They are the glioma-associated protein SETA To whom correspondence should be addressed. Tel.: 81-52-789-4088; Fax: 81-52-789-5542; E-mail: [email protected]. (21), which is one of the isoforms of CIN85/Ruk (22, 23), and 39104 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. CHMP4b as Alix-interacting Protein 39105 cDNA fragments obtained by digestion with restriction enzymes were endophilins (10). These proteins, including ALG-2, associate inserted into pEGFP-C2 or pEGFP-C3 (Clontech) vectors. To delete the with the proline-rich region in Alix by Src homology 3 domains putative 5-untranslated region of CHMP4b by the PCR method, the in the case of SETA and endophilins or by a penta-EF-hand forward primer containing a translational initiation codon (underlined), domain in the case of ALG-2. Conservation among the homo- 5-GCGAATTCCATGTCGGTGTTCGGGAAG-3, and the reverse logues, however, is lower within their C-terminal proline-rich primer containing the stop codon (underlined), 5-GCCTCGAGCCATT- regions, and yeast Rim20p lacks this region. In contrast, the ACATGGATCCAGCCCA-3, were designed on the basis of the CHMP4b cDNA sequence. The PCR product was cloned into a pKF3 (TAKARA N-terminal parts of Alix homologues show strong similarities, Shuzo, Kyoto, Japan). To create a pCMV-3FLAG vector for eukaryotic particularly in the regions containing a consensus site of po- expression, forward oligonucleotides, 5-GATCGACTACAAAGACCAT- tential tyrosine phosphorylation by Src family tyrosine ki- GACATCGATTATAAGGATGACGACGAT-3 and 5-GGGATCCCTGC- nases. We speculated that the N-terminal region of Alix plays A-3, and reverse oligonucleotides, 5-TCATCCTTATAATCGATGTCA- an important role in functions common to all Alix homologues. TGGTCTTTGTAGTC-3 and 5-GGGATCCCATCG-3, were inserted In this study, we screened for proteins binding to the N- between BamHI and PstI sites of the pCMV-Tag2C vector (Stratagene). A fragment of the full-length CHMP4b cDNA was inserted into a terminal region of human Alix by the yeast two-hybrid method, pCMV-3FLAG vector and a pEGFP-N1 vector (Clontech), and the and we isolated two clones encoding proteins that are similar to resultant plasmids, designated pFLAG-CHMP4b and pCHMP4b-GFP, each other. One is identical to HSPC134/CHMP4 (chromatin- were used to express 3FLAG-tagged protein and GFP fusion protein, modifying protein; charged multivesicular body protein) (24, respectively. The plasmid encoding GFP-LC3 protein was constructed E235Q 25), which is designated as CHMP4a in this paper, and the as described previously (30). A Myc-SKD1 fragment from pEGFP- EQ other is a novel protein designated as CHMP4b. These CHMP4 MSKD1 (31) was inserted into the EcoRI site of a pCI-neo vector E235Q (Promega) to generate pMyc-SKD1 . proteins are highly homologous to yeast Snf7/Vps32 (vacuolar Yeast Two-hybrid Screening—The MATCHMAKER two-hybrid sys- protein sorting) (26, 27). Yeast Snf7/Vps32 is a member of class tem, including yeast strains and vectors, was obtained from Clontech, E Vps proteins, which are required for sorting of membrane and the screening was performed as described previously (6). Positive proteins into inner vesicles that originate by inward invagina- clones were sequenced using an automated fluorescent sequencer, ABI tion in the lumen of the late endosome (multivesicular body PRISM 310 (PE Applied Biosystems). A computer homology search (MVB) ) (28). Subcellular localization of CHMP4b and the ef- using the advanced BLAST program and coiled-coil region prediction were performed on the World Wide Web. Phylogenetic analysis using fect of CHMP4b overexpression were analyzed, and the ob- Clustal X was performed as described previously (32). tained results suggest that Alix and CHMP4b participate in the Cell Culture and Transfection—HEK293 and HeLa cells were cul- endosomal sorting pathway. tured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), and EXPERIMENTAL PROCEDURES streptomycin (100 g/ml) at 37 °C under humidified air containing 5% Antibodies—Mouse monoclonal antibodies were purchased: EEA1 CO . One day after HEK293 cells or HeLa cells had been seeded, the (early endosome antigen 1; Transduction Laboratories), Lamp-1 (lyso- cells were transfected with the expression plasmid DNAs by the con- somal membrane protein-1)/CD107a (Pharmingen), multiubiquitin ventional calcium phosphate precipitation method or by using Fu- (FK2) (MBL), green fluorescent protein (GFP) (Santa Cruz Biotechnol- GENE6 (Roche Applied Science). After 24 h, cells were harvested and ogy, Inc., Santa Cruz, CA), FLAG tag (M2), c-Myc (9E10) (Sigma), and analyzed. To generate HEK293 stable transfectants that constitutively V5 tag (Invitrogen). Rabbit polyclonal antibodies against FLAG tag and express 3FLAG-tagged CHMP4b (FLAG-CHMP4b/HEK293), GFP were purchased from Sigma and Abcam, respectively. Anti-cation- HEK293 cells transfected with the expression vector (pFLAG-CHMP4b) independent mannose 6-phosphate receptor (anti-CI-MPR) antibody were selected by culturing in the presence of 0.5 mg/ml of G418 for was prepared as described previously (29). Peroxidase-conjugated goat 2 weeks. anti-rabbit and anti-mouse IgG antibodies were from Wako (Osaka, Expression and Purification of GST-AlixC—Escherichia coli BL21 Japan). Cy3-labeled anti-rabbit and anti-mouse antibodies and fluores- cells were transformed with the plasmid pGST-AlixC. The GST fusion cein isothiocyanate-conjugated anti-mouse antibodies used for indirect protein was purified by binding to glutathione-Sepharose 4B (Amer- immunofluorescence analyses were purchased from Amersham Bio- sham Biosciences) according to the manufacturer’s instructions. GST- sciences and Rockland, respectively. AlixC was eluted from Sepharose beads with 10 mM reduced glutathi- Plasmid Constructions—Human Alix cDNA was cloned from Human one in 50 mM Tris-HCl, pH 8.5, dialyzed against 50 mM Tris-HCl, pH TM Fetus Marathon-Ready cDNA (Clontech) by the PCR method and 7.5, and then stored at 4 °C until use. inserted into a pCR2.1-TOPO vector (Invitrogen). pCR2.1-TOPO-Alix Pull-down Assay—One day after FLAG-CHMP4b/HEK293 (6  10 ) Y319F, which has a point mutation at amino acid 319 (from tyrosine to cells had been seeded, the cells were harvested and washed with PBS phenylalanine), was created by PCR-based site-directed mutagenesis (137 mM NaCl, 2.7 mM KCl, 8 mM Na HPO , 1.5 mM KH PO , pH 7.3) 2 4 2 4 according to the provided instruction for QuickChange site-directed and then lysed in 450 l of lysis buffer (10 mM HEPES-NaOH, pH 7.4, mutagenesis kit from Stratagene using two complementary primers 142.5 mM KCl, 0.2% Nonidet P-40, 0.1 mM pefabloc, 25 g/ml leupeptin, (5-AATGACTTCATTTTTCATGATCGAGTT-3 and 5-AACTCGAT- 1 M E-64, and 1 M pepstatin). Supernatants (each 200 l, obtained by CATGAAAAATGAAGTCATT-3), and the mutation was confirmed by centrifugation at 10,000  g) were incubated with 10 g of GST or DNA sequencing. To obtain the cDNA fragment encoding amino acids GST-AlixC protein that had been immobilized on glutathione-Sepha- 1– 660 (AlixC), the palindromic universal translational terminator, rose for3hat4 °C. After complexes had been washed three times with 5-GCTTAATTAATTAAGC-3, was inserted into the blunt-ended NdeI the lysis buffer, they were subjected to Western blotting analysis using site of the Alix cDNA. A cDNA fragment corresponding to AlixC was anti-FLAG monoclonal antibody (mAb). Signals were detected by either inserted into the yeast expression vector pGBKT7 (Clontech), the glu- the chemiluminescent method using Super Signal West Pico Chemilu- tathione S-transferase (GST) fusion vector pGEX-4T-2 (Amersham Bio- minescent Substrate (Pierce) or diaminobenzidine staining. sciences), and a mammalian expression vector pcDNA6/V5-His (Invitro- Immunoprecipitation—FLAG-CHMP4b/HEK293 cells transfected gen) to construct pGBKT7-AlixC, pGST-AlixC, and pAlixC-V5, with the expression vector of either AlixC-V5, GFP-Alix mutants, or respectively. Using the SmaI site located upstream of the stop codon in Myc-SKD1 were lysed in the lysis buffer and then subjected to centrif- the Alix cDNA, a cDNA fragment corresponding to amino acid residues ugation at 10,000  g. The supernatants were incubated with anti-V5 1– 847 was inserted into pcDNA6/V5-His to construct pAlix-V5. To mAb, anti-GFP pAb, or anti-FLAG mAb for 1 h and then incubated for generate truncated forms of GFP-Alix fusion proteins, various Alix 1 h after the addition of Protein G-Sepharose 4 Fast Flow (Amersham Biosciences). The Sepharose beads were then washed with the lysis buffer three times and subjected to Western blotting analysis using 1 either anti-V5 mAb, anti-FLAG mAb, anti-GFP mAb, or anti-Myc mAb. The abbreviations used are: MVB, multivesicular body; CI-MPR, Immunofluorescent Staining—HeLa or HEK293 cells grown on cov- cation-independent mannose 6-phosphate receptor; EGF, epidermal erslips were fixed in 4% paraformaldehyde/PBS and permeabilized in growth factor; ESCRT, endosomal sorting complex required for trans- 0.1% Triton X-100/PBS. After blocking with 0.1% gelatin/PBS, the cells port; GFP, green fluorescent protein; GST, glutathione S-transferase; were incubated with primary antibodies either at 4 °C overnight or at HEK, human embryonic kidney; LC3, microtubule-associated protein 1 room temperature for 1 h and then with secondary antibodies at room light chain 3; mAb, monoclonal antibody; PBS, phosphate-buffered sa- line; pAb, polyclonal antibody; E3, ubiquitin-protein isopeptide ligase. temperature for 1 h. Finally, they were mounted with antifading solu- 39106 CHMP4b as Alix-interacting Protein tion (25 mM Tris-HCl (pH 8.7), 10% polyvinyl alcohol, 5% glycerol, 2.5% region of Alix was essential but not sufficient for CHMP4b 1,4-diazobicyclo-(2,2,2)-octane). Immunofluorescences were analyzed interaction. Since the Tyr of Alix is highly conserved among under a confocal laser-scanning microscope, LSM5 PASCAL (Zeiss). Alix homologues in various organisms and serves as a potential Epidermal Growth Factor (EGF) Uptake Assay—The transfected phosphorylation site for Src-type tyrosine kinases, we replaced HeLa cells were incubated with Dulbecco’s modified Eagle’s medium for Tyr of AlixC with phenylalanine to examine whether this 1hat37 °C and then with 0.5 g/ml tetramethylrhodamine-EGF (Mo- lecular Probes, Inc., Eugene, OR) in 0.5 mg/ml bovine serum albumin/ putative phosphorylation site is needed for the interaction. The Dulbecco’s modified Eagle’s medium for 1 h at 4 °C. The cells were interaction between CHMP4b and Y319FC was still detecta- washed and incubated in 10% fetal bovine serum/Dulbecco’s modified ble with a slight decrease in signal. Eagle’s medium for 30 min or6hat37 °C. Fluorescence Microscopic Analysis of Overexpressed CHMP4b—We investigated the subcellular localization of RESULTS CHMP4b using HeLa cells transfected with pFLAG-CHMP4b. Yeast Two-hybrid Screening—To search for Alix-interacting Double immunofluorescent staining was performed using an proteins, the N-terminal region (amino acids 1– 660) of human anti-FLAG antibody (Fig. 4, A, E, I, and M) and either an Alix (AlixC) that was fused to the Gal4 DNA binding domain antibody of protein that is a marker of early endosome (EEA1) was used as bait, and a HeLa cell cDNA library was used as (Fig. 4B), late endosome and lysosome (Lamp-1) (Fig. 4F), or prey in a yeast two-hybrid screen system. Sixty-two positive the trans-Golgi network and late endosome (CI-MPR) (Fig. 4J). clones were isolated from 6  10 transformants. A search of We also used GFP-LC3 as an autophagosomal membrane DNA data bases revealed that the positive clones corresponded marker instead of staining with an antibody (Fig. 4N). We to 12 different proteins. Two of them were presumed to be found that FLAG-CHMP4b was distributed in a punctate man- human homologues of yeast Snf7/Vps32 (26, 27) (Fig. 1), and ner mainly in the perinuclear area (Fig. 4, A, E, I, and M) and they were also similar to each other (60.7% identity; 136 of 224 slightly diffused in the cytoplasm. However, only the diffuse amino acid residues). One was identical to HSPC134 (24), pattern was observed in some cells (data not shown). As shown which was referred to as CHMP4 by Howard et al. (25), and the in merged images of the same panels (Fig. 4, C, G, and K) and other was very similar to a hypothetical protein registered in in images of higher magnification (Fig. 4, D, H, and L), the TM the sequence data bases (DDBJ/GenBank /EMBL accession distribution of FLAG-CHMP4b partly overlapped in the pe- number AL050349). In this paper, we designated HSPC134/ rinuclear region with the distributions of EEA1 and Lamp-1, CHMP4 as CHMP4a and the other as CHMP4b, respectively. respectively, in the pFLAG-CHMP4b-transfected cell (Fig. 4, C, Both CHMP4a and CHMP4b were predicted to contain coiled- left cell, and G, bottom right cell), and the degree of overlapping coil regions (Fig. 1A). Their N-terminal regions are rich in basic was greater than that of CI-MPR (Fig. 4K, center cell). On the residues, whereas their C-terminal regions are acidic. The other hand, GFP-LC3 showed both punctate and diffuse pat- CHMP4 proteins are part of a large CHMP family (25). The terns (Fig. 4N, inset), and its fluorescent signals were also data base search revealed an additional CHMP4 homologue in detected in the nucleus under the present conditions, in which this subfamily in the human genome (Fig. 1B). autophagy was not enhanced by subjecting the cells to nutrient GST Pull-down Assays of AlixC and CHMP4b—We per- starvation. The distribution of GFP-LC3 was not noticeably formed a GST pull-down assay to determine whether the changed by overexpression of FLAG-CHMP4b (Fig. 4N) and did CHMP4b protein from mammalian cells has the capacity to not overlap with that of FLAG-CHMP4b in cells expressing bind to Alix. First, we constructed several plasmids for express- both GFP-LC3 and FLAG-CHMP4b (Fig. 4, O and P). ing various fusion proteins and generated HEK293 cell lines To determine whether the distribution of ubiquitinated pro- constitutively expressing FLAG-CHMP4b (FLAG-CHMP4b/ teins was affected by CHMP4b overexpression, we examined HEK293). After incubation of the lysate of FLAG-CHMP4b/ the localization of ubiquitinated proteins by staining HeLa HEK293 with the GST fusion protein of AlixC (GST-AlixC) cells transfected with pCHMP4b-GFP using anti-ubiquitin that had been immobilized on glutathione-Sepharose beads, mAb (FK2) that recognizes mono- and polyubiquitinated pro- the proteins bound to the beads were subjected to Western teins but not free monoubiquitin (33, 34). In most of the trans- blotting using an anti-FLAG mAb as a probe. Whereas fected cells, the distribution of CHMP4b-GFP was essentially CHMP4b did not bind to the negative control GST beads, it similar to that of FLAG-CHMP4b (Figs. 4 and 5A). As shown in bound to the GST-AlixC beads (Fig. 2A). Fig. 5B, fluorescent signals of ubiquitinated proteins became Co-immunoprecipitation of CHMP4b and AlixC—When the stronger in cells expressing CHMP4b-GFP, particularly in the lysate of FLAG-CHMP4b/HEK293 cells transfected with the perinuclear area, and showed punctate patterns. Furthermore, expression vector of V5-tagged AlixC (pAlixC-V5) was im- ubiquitinated proteins in this area were partially co-localized munoprecipitated with anti-FLAG mAb, the precipitates con- with CHMP4b-GFP (Fig. 5, C and D). When exogenously ex- tained AlixC-V5 as revealed by Western blotting using an- pressed FLAG-tagged ubiquitin was monitored, accumulation ti-V5 mAb (Fig. 2B). Moreover, in a reciprocal experiment, and partial co-localization of FLAG-tagged ubiquitin with GFP- FLAG-CHMP4b was also co-precipitated with AlixC-V5 using tagged CHMP4b were similarly observed (data not shown). anti-V5 mAb for immunoprecipitation (data not shown). Accumulation of ubiquitinated proteins in the cells overex- Co-immunoprecipitation of CHMP4b with Alix Segments— pressing CHMP4b suggests that overexpression of CHMP4b To narrow the CHMP4b binding region in AlixC, we per- may disturb the endosomal sorting pathway. To provide func- formed co-immunoprecipitation analysis of FLAG-CHMP4b and GFP fusions of full-length or various truncated forms of tional data supporting this speculation, we monitored the fate of endocytosed EGF. HeLa cells transfected with pCHMP4b- Alix. The lysates of FLAG-CHMP4b/HEK293 cells transfected with the expression vectors of GFP-Alix mutants were immu- GFP were incubated with Tetramethylrhodamine-EGF at 4 °C for 1 h, washed, and then allowed to uptake EGF at 37 °C for 30 noprecipitated with anti-GFP pAb, and the precipitates were subjected to Western blotting. As shown in Fig. 3, A and B, and min or 6 h. Internalized EGF was observed at 30 min, and summarized in Fig. 3C, we observed interaction of FLAG- noticeable change was not detected between the CHMP4b-GFP CHMP4b with Alix mutants 1– 868, 1– 660, and 1– 423 and expressing cells and the untransfected cells (Fig. 5, E–G). At weaker interaction with Alix mutant 115– 660. No interaction 6 h, fluorescent signals of tetramethylrhodamine-EGF were was detected with Alix mutants 1–329, 221– 660, and 329 – 660. undetectable in most of the untransfected cells, whereas EGF Bro1-rhophilin conserved domain located in the N-terminal remained in the perinuclear region of the cells expressing CHMP4b as Alix-interacting Protein 39107 FIG.1. Primary structure comparison of CHMP4 proteins with Snf7 and other CHMP family members. A, amino acid sequence alignment of CHMP4 proteins and Snf7. Identical residues between CHMP4 and Snf7 are stippled. The amino acid sequence of Snf7 is from TM GenBank /EMBL/DDBJ (Saccharomyces cerevisiae Snf7; P39929). The coiled-coil regions predicted by the COILS program are indicated with bars above the sequence of CHMP4b. B, phylogenetic comparison of CHMP4 subfamily. Using the default setting of Clustal X 1.81, the amino acid sequences were aligned, and a bootstrap tree file was created. The phylogenetic tree was drawn with NJplot. Horizontal branch lengths are drawn TM to scale with the bar indicating 0.1 amino acid replacement/site. A hypothetical protein is indicated by GenBank /EMBL/DDBJ accession number. Chromosomal loci in the human genome are indicated. h, human; Sc, S. cerevisiae. CHMP4b-GFP (Fig. 5, H–J). However, there was a variation in tially concentrated in the perinuclear region of the Alix-V5 the amount of remaining EGF among the CHMP4b-GFP trans- transfectants (Fig. 6B). In contrast, in the co-transfected cells, fectants (data not shown). Alix-V5 was distributed in the perinuclear region (Fig. 6E), and Change in Alix Distribution by Overexpression of most of the Alix was co-localized with CHMP4b (Fig. 6, D and CHMP4b—To determine whether CHMP4b affects the subcel- F). A similar effect was observed using the pAlixC-V5 con- lular localization of Alix, we performed immunostaining of struct (data not shown). HeLa cells transfected with pAlix-V5 (which encodes V5-tagged Formation of Vesicle-like Structures—As shown in Fig. 7A, Alix) alone or both pAlix-V5 and pFLAG-CHMP4b (Fig. 6). unlike obvious punctate patterns of localization of FLAG- Alix-V5 was diffusely distributed in the cytoplasm and par- CHMP4b in transiently overexpressed HeLa cells (Fig. 4) and 39108 CHMP4b as Alix-interacting Protein E235Q compartment in the cells expressing SKD1 , a dominant negative form of SKD1. To examine whether CHMP4b also interacts with SKD1 and accumulates in the E235Q compart- ment, we performed immunoprecipitation experiments. The lysate of FLAG-CHMP4b/HEK293 cells transfected with the E235Q expression vectors of either Myc-SKD1 or Myc-SKD1 was immunoprecipitated with anti-FLAG mAb, and the precipi- tates were subjected to Western blotting. As shown in Fig. 8A, whereas the interaction of Myc-SKD1 with FLAG-CHMP4b was not detectable under the condition used, the interaction of E235Q Myc-SKD1 with FLAG-CHMP4b was detected. We then performed immunostaining of HeLa cells transfected with ex- E235Q pression vectors of Myc-SKD1 and CHMP4b-GFP. As shown in Fig. 8, B–D, the distribution of CHMP4b-GFP was E235Q very similar to that of Myc-SKD1 , and there were consid- erable overlaps between their distributions. To investigate whether Alix also accumulates in the E235Q compartment, we further performed immunostaining using HeLa cells express- E235Q ing both Myc-SKD1 and GFP-Alix. The accumulation of a fraction of GFP-Alix was observed, and the increased GFP-Alix E235Q signals overlapped those of Myc-SKD1 (Fig. 8, E–G). DISCUSSION By a yeast two-hybrid screen using AlixC (amino acids 1– 660) as bait, we isolated two clones, which are similar to each other, as novel Alix-interacting proteins. By data base analysis, one of them was found to be identical to HSPC134, whose cDNA was isolated as one of the 300 previously unde- fined genes expressed in CD34 hematopoietic stem/progenitor cells (24), and HSPC134 was designated as CHMP4 by Howard et al. (25). In this paper, we designated HSPC134/CHMP4 as CHMP4a and the other similar protein as CHMP4b. These two proteins are homologues of yeast Snf7/Vps32 (Fig. 1), which is one of the class E Vps proteins and is essential for vacuolar FIG.2. Interaction between CHMP4b and AlixC. A, the lysate protein sorting and transport (28). In addition to CHMP4a and of HEK293 cells constitutively expressing FLAG-CHMP4b was incu- CHMP4b, we found another CHMP4 homologue in the human bated with glutathione-Sepharose beads that carried GST or GST- genome data base (Fig. 1B). Their expression patterns in tis- AlixC, and the beads then were pelleted by centrifugation. The pellets were analyzed by Western blotting with anti-FLAG mAb. B, HEK293 sues and during development need to be analyzed in cells constitutively expressing FLAG-CHMP4b were transfected with future studies. pAlixC-V5. After 24 h, transfectants were lysed and immunoprecipi- In general, coiled-coil regions mediate the protein-protein tated (IP) with anti-FLAG mAb (FLAG) or control mouse IgG (con). The interactions. Since both Alix and CHMP4b have coiled-coil immunoprecipitates were analyzed by Western blotting (WB) with an- regions, at first we speculated that these regions were essential ti-V5 mAb or anti-FLAG mAb. Mouse immunoglobulin heavy chains (IgG-H) and light chains (IgG-L) in the immunoprecipitates were also for the interaction between the two proteins. However, as detected by subsequent Western blotting using peroxidase-conjugated shown in Fig. 3, the coiled-coil regions in Alix were not required anti-mouse IgG as a secondary antibody. The molecular masses of for their interaction, and the determined CHMP4b-interacting standard proteins are indicated on the left. region possesses a potential phosphorylation site by Src-type tyrosine kinases. Indeed, substitution of Tyr with Phe in HEK293 cells (data not shown), FLAG-CHMP4b showed (Y319F mutant) has been shown to lose the ability to interact diffuse cytoplasmic distribution in stably expressing cells with both focal adhesion kinase and PYK-2 (35). The Y319F (FLAG-CHMP4b/HEK293), but vesicle-like structures were ob- mutant, however, could substantially interact with CHMP4b served in 5% of the cells (data not shown). On the other hand, (Fig. 3A). Xu et al. (18) reported that Rim20p, which is another Alix-V5 and AlixC-V5 were localized in the cytoplasm, even in yeast homologue of Alix, associates with Rim101p regardless of HEK293 cells transiently transfected with pAlix-V5 (Fig. 7B) substitutions of Ile-Tyr to Ala-Ala in this highly conserved or pAlixC-V5 (Fig. 7C). In the FLAG-CHMP4b/HEK293 cells region. Although Tyr in human Alix is widely conserved transfected with pAlixC-V5, FLAG-CHMP4b and AlixC-V5 from lower to higher eukaryotic cells, the importance of Tyr showed punctate patterns of distribution (Fig. 7, G, H, J, and for protein complex formation appears to be dependent on K) in 94% of the cells examined, and these two proteins were interacting partners. co-localized in vesicle-like structures (Fig. 7, I and L, indicated In yeast cells, class E Vps proteins are essential for carboxyl by the arrowheads). In contrast, the effect of overexpression of peptidase S sorting, and a group of class E Vps proteins form Alix-V5 in FLAG-CHMP4b/HEK293 cells on CHMP4b localiza- three complexes, named ESCRT-I, -II, and -III (endosomal tion was not significant (Fig. 7, D, E, and F), and vesicle-like sorting complex required for transport) (36 –38). ESCRTs work structures stained with FLAG-CHMP4b and Alix-V5 were ob- in the sorting of ubiquitinated proteins in the process of the served in only 14% of the cells examined (data not shown). invaginating endosomal membrane toward the lumen, and this Accumulation of CHMP4b and Alix at the E235Q Compart- compartment containing inner vesicles is called the MVB. ES- ment—Howard et al. (25) reported that CHMP1 interacts with CRT-III is composed of four similar yeast CHMP family pro- SKD1, which is an AAA-type ATPase homologous to yeast teins, among which Snf7 forms a subcomplex with Vps20 and Vps4, and that CHMP1 accumulates in the so-called E235Q Vps2 forms a subcomplex with Vps24. It has recently been CHMP4b as Alix-interacting Protein 39109 FIG.3. Immunoprecipitation of CHMP4b with Alix truncated mutants. A and B, HEK293 cells constitutively expressing FLAG-CHMP4b were transfected with the expression vectors for various GFP-Alix truncated mutants. After 24 h, transfectants were lysed and immunoprecipitated with anti-GFP pAb. The immunoprecipitates were analyzed by Western blotting (WB) with anti-GFP mAb or anti-FLAG mAb. The numbers indicate the N-terminal and C-terminal Alix residues of the truncation mutants. C, a schematic representation of Alix truncated mutants is shown. The numbers indicate the N-terminal and C-terminal Alix residues of mutants. Y, the tyrosine residue in a highly conserved potential recognition site for Src type tyrosine kinases. F, the substituted phenylalanine residue by the point mutation. The relative strengths of the interactions are indicated as follows. , equivalent to full-length; , slightly weak; , weak; , not detectable. BRD, Bro1-rhophilin conserved domain (NCBI conserved domain data base accession number 17278; pfam03097); CC, coiled-coil; PRR, proline-rich region. reported that Bro1 (yeast homologue of Alix) is identical to cells, the amount of the FLAG-CHMP4b protein in the tran- Vps31, which is also a class E Vps protein (28, 39). Further, the siently expressed HEK293 cells was estimated about 1.5–2 endosomal association of Bro1 has been shown to be specifically times as much as the amount of the protein expressed in the dependent on Snf7 (40). Although direct interaction between FLAG-CHMP4b/HEK293 cells (data not shown). Considering Bro1/Vps31 and Snf7/Vps32 has not been demonstrated, the the transfection efficiency of 25–50%, the FLAG-CHMP4b pro- association of these proteins was suggested by results of sys- tein in an individual transient transfectant was calculated to tematic analysis of protein complexes by mass spectrometry be present 3– 8 times more per cell than in the stably express- (41). Moreover, the interaction between another yeast Alix ing cells. Thus, the punctate subcellular localization of exog- homologue, Rim20p, and Snf7 was also suggested by results of enously expressed CHMP4b may depend on the expression genome-wide two-hybrid analysis (42). Since both CHMP4 and levels but not on the types of the cells. Indeed, diffuse cytoplas- Alix are widely present from lower to higher eukaryotic cells, mic distributions were also observed in some HeLa cells by the function mediated by the interaction between the two pro- transient expression of the N-terminally tagged proteins teins is presumed to be conserved and seems to be involved in (FLAG-CHMP4b and GFP-CHMP4b), particularly, using the regulation of sorting in the endosomal pathway. lesser amounts of expression vectors (data not shown). Since In HeLa cells (Figs. 4 and 5) and also in HEK293 cells (data the C-terminally tagged CHMP4b-GFP protein is more prone not shown), most of the transiently overexpressed CHMP4b to exhibit cytoplasmic punctate distributions under the similar proteins are distributed in punctate patterns mainly in the transfection condition, tagging at the N terminus of CHMP4b perinuclear area. On the other hand, in HEK293 cells stably might affect its stability, folding, and/or interaction with other expressing FLAG-CHMP4b (FLAG-CHMP4b/HEK293), the proteins. diffuse distribution of CHMP4b in the cytoplasm was observed As in the yeast system, where the dissociation of Snf7 from (Fig. 7A). By Western blot analysis using similar numbers of the limiting membrane of MVB has been shown to depend on 39110 CHMP4b as Alix-interacting Protein FIG.6. Effect of FLAG-CHMP4b overexpression on the distri- bution of Alix-V5. HeLa cells transfected with the pAlix-V5 alone (A–C) or both pAlix-V5 and pFLAG-CHMP4b (D--F) were subjected to immunofluorescence confocal microscopy using anti-FLAG pAb (A and D) and anti-V5 mAb (B and E). A and D, FLAG-CHMP4b; B and E, Alix-V5; C and F, merged images of either A and B or D and E. Bar, 10 m. FIG.4. Subcelluar localization of CHMP4b in HeLa cells. HeLa cells transfected with pFLAG-CHMP4b alone (A–L) or both pFLAG- CHMP4b and pEGFP-C1-LC3 (M–P) were subjected to immunofluores- cence confocal microscopy using polyclonal (A, E, and M) or monoclonal (I) anti-FLAG antibodies and anti-endosomal marker protein antibod- ies (anti-EEA1 mAb, anti-Lamp1 mAb, or anti-CI-MPR pAb). A, E, I, and M, immunofluorescent micrographs of FLAG-CHMP4b; B, EEA1; F, Lamp-1; J, CI-MPR; N, fluorescent micrographs of GFP-LC3; C, G, K, and O, merged images; D, H, L, and P, higher magnifications of images shown in C, G, K, and O; insets in M and N, the cells expressing GFP-LC3 alone in a different field. Bar,10 m. FIG.7. Formation of vesicle-like structures and CHMP4b ac- cumulation by AlixC overexpression. HEK293 cells (B and C)or FIG.5. Effects of CHMP4b overexpression on the endocytic HEK293 cells constitutively expressing FLAG-CHMP4b (FLAG- pathway. A–D, HeLa cells transfected with pCHMP4b-GFP were CHMP4b/HEK293) (A and D–L) transfected with pAlix-V5 (B and D–F) stained with anti-ubiquitin mAb (FK2). A, CHMP4b-GFP; B, ubiquitin; or pAlixC-V5 (C and G–L) were subjected to immunofluorescence C, merged image of A and B; D, higher magnification of the image confocal microscopy using anti-FLAG pAb (A, D, and G) and anti-V5 shown in C. E–J, HeLa cells transfected with CHMP4b-GFP were mAb (B, C, E, and H). F and I, merged images of either G and H or J and incubated at 4 °Cfor1hinthe presence of tetramethylrhodamine-EGF, K. J–L, higher magnification of images shown in G–I, respectively. The washed, and then incubated at 37 °C for 30 min (E–G)or6h(H–J). E arrowheads indicate the vesicle-like structures. Bar,10 m. and H, CHMP4b-GFP; F and I, tetramethylrhodamine-EGF; G and J, merged images. The asterisks indicate untransfected cells. Bars in C, G, and J,10 m; D,2 m. E235Q tion. CHMP4b was co-immunoprecipitated with SKD1 , the ATPase activity of Vps4 (27), the localization of CHMP4b which is a SKD1 mutant defective in ATP hydrolysis, but not may be also regulated by SKD1, a mammalian homologue of with wild type SKD1 (Fig. 8A). This result agrees with the Vps4 (43, 44). In the present study, this hypothesis was exam- previous report that blocking ATP hydrolysis of an AAA type ined first by analysis of the interaction between CHMP4b and ATPase by mutation (Glu to Gln mutation in the Walker B box) SKD1 and then by analysis of their intracellular co-localiza- results in high affinity interactions of the AAA protein with its CHMP4b as Alix-interacting Protein 39111 E235Q E235Q FIG.8. Interaction between CHMP4b and SKD1 and co-localization of CHMP4b and Alix with SKD1 . A, HEK293 cells E235Q constitutively expressing FLAG-CHMP4b were transfected with either pMyc-SKD1 or pMyc-SKD1 . After 24 h, transfectants were lysed and immunoprecipitated (IP) with anti-FLAG mAb (FLAG) or control mouse IgG (con). The immunoprecipitates were analyzed by Western blotting (WB) with anti-FLAG mAb or anti-Myc mAb. IgG-H and IgG-L indicate mouse immunoglobulin heavy chains and light chains, respectively. B–G, E235Q HeLa cells co-transfected with the expression vectors of Myc-SKD1 and either CHMP4b-GFP (B–D) or GFP-Alix (E–G) were subjected to E235Q immunofluorescence confocal microscopy using anti-Myc mAb. B, CHMP4b-GFP; E, GFP-Alix; C and F, Myc-SKD1 ; D and G, merged images of either B and C or E and F. Bar,10 m. protein targets (45). We observed the co-localization of CHMP4b was partially co-localized with EEA1 and Lamp-1 E235Q CHMP4b with SKD1 in HeLa cells expressing CHMP4b- and to a lesser degree with CI-MPR (a marker protein for late E235Q GFP and Myc-SKD1 (Fig. 8, B–D). Furthermore, the co- endosome and trans-Golgi network), suggesting localization of localization of CHMP4b-GFP with endogenous SKD1 was ob- CHMP4b from early to late endosomes. Ubiquitinated proteins served using anti-SKD1 antibody (data not shown). Since accumulated in cells overexpressing CHMP4b, and CHMP4b excessive CHMP4b in the transiently expressed cells may se- was partly co-localized with ubiquitinated proteins like other quester endogenous SKD1, CHMP4b molecules may not be able endosomal marker proteins (Fig. 5, A–D). The electrophoretic to dissociate from the endosomal membranes and may keep mobilities of the FLAG-CHMP4b proteins analyzed by Western associating with other ESCRT components. The accumulation blotting using anti-FLAG antibody were not different between of Alix in the E235Q compartment was observed as in the case the lysates prepared from diffusely stained cells (FLAG- of CHMP4b (Fig. 8, E–G). This result suggests that the disso- CHMP4b/HEK293) and those from transiently overexpressed ciation of Alix from endosomes might also depend on the HeLa cells that exhibited punctate distributions, and no higher ATPase activity of SKD1. molecular mass FLAG-CHMP4b proteins indicative of ubiquiti- The distributions of endosomal marker proteins such as nation were observed in either cell lysates by Western blotting EEA1 (early endosome) and Lamp-1 (late endosome and lyso- (data not shown). Thus, the observed co-localization of the some) were affected by overexpression of CHMP4b (Fig. 4). ubiquitinated proteins and CHMP4b-GFP shown in Fig. 5, 39112 CHMP4b as Alix-interacting Protein A–D, may not be due to ubiquitination of the CHMP4 protein have an important role for dissociation from endosomal mem- itself. Ubiquitination of proteins is not only a signal of protea- branes. Our recently obtained data indicate that the binding somal degradation of cellular proteins but also a triggering sites for ALG-2, CIN85, and endophilin are all located at three signal for sorting of plasma membrane proteins such as growth different sites within the C-terminal proline-rich region. Therefore, association or dissociation of Alix with these factors factor receptors to lysosomal degradation through the endoso- mal sorting pathway, where Hrs and TSG101 play roles in may trigger a conformational change of Alix and may affect the ability of Alix to interact with CHMP4b. Interestingly, CIN85 directing ubiquitinated proteins via their ubiquitin-binding do- and endophilin are also factors involved in membrane receptor mains to the components of MVB sorting on endosomal mem- internalization triggered by Cbl, which has intrinsic ubiquiti- branes (28, 46). Thus, the accumulation of the ubiquitinated nation activities as a RING-type E3 ligase (47, 48). proteins (Fig. 5, A–D) and the finding that overexpression of Springael et al. (39) reported that Npi3, which is identical to CHMP4b affected the distribution of endosomal markers (Fig. Bro1, is required for vacuolar sorting of Gap1 permease under 4) suggest disturbance of the endosomal protein sorting path- ubiquitin-dependent control in budding yeast. Compared with way by overexpressed CHMP4b proteins. To provide experi- the genetically well studied yeast system, information on the mental evidence in favor of this speculation, we monitored a mammalian MVB sorting pathway is still limited. Recent data, fate of endocytosed EGF. Disappearance of the fluorescent sig- however, indicate that factors involving ubiquitin actions in the nals of labeled EGF was inhibited in the HeLa cells transfected pathway are conserved between yeast and mammals (e.g. with CHMP4b-GFP (Fig. 5, H–J), raising a possibility that Vps27 versus Hrs, Vps23 versus TSG101, and Rsp5 versus overexpression of CHMP4b disturbs the regulation of mem- Nedd4) (28, 49). Interestingly, TSG101 was isolated as a can- brane trafficking from endosomes to lysosomes. didate cDNA clone of the Alix-interacting partner using a frag- Overexpression of CHMP4b caused accumulation of Alix ment containing the proline-rich region of Alix as bait by the from the cytoplasm to the perinuclear area, where CHMP4b yeast two-hybrid screen. Alix might link components of ubiq- was co-localized (Fig. 6). We also found that overexpression of uitin-dependent internalization machinery to components of AlixC-V5 induced formation of vesicle-like structures in cells the MVB sorting complexes by interacting with multiple stably expressing FLAG-CHMP4b (FLAG-CHMP4b/HEK293) factors. (Fig. 7, G–L). These findings suggest that Alix cooperates with Although the exogenously expressed FLAG-CHMP4b pro- CHMP4b and participates in the endosomal pathway. Since no teins exhibited cytoplasmic punctate patterns, they were also vesicular structures were observed in HEK293 cells transfected observed in the nucleus in some cells (Figs. 4 and 7). CHMP1, with pAlixC-V5 (Fig. 7C), the level of endogenous CHMP4b which is implicated in MVB formation (25), was first found as seems to be too low to induce vesicle formation. In contrast, a partner of Polycomb group protein Pcl (Polycomblike) by a transient overexpression of CHMP4b both in HeLa cells (Figs. yeast two-hybrid screen, and a role in stable gene silencing 4 and 5) and in HEK293 cells (data not shown) induced the within the nucleus was suggested (50). A post-translationally accumulation of CHMP4b in the perinuclear area as punctate modified form of CHMP1 is associated with nuclear matrix, patterns without exogenous Alix, suggesting a sufficient ex- and overexpressed CHMP1 localizes to a punctate subnuclear pression level of endogenous Alix for CHMP4b interaction. pattern and arrests cells in S-phase (50). On the other hand, Previously, Chatellard-Causse et al. (10) reported that overex- some mammalian class E Vps homologues were identified as pression of Alix-CT, the C-terminal half of Alix, led to cytoplas- components of nuclear factors. For instance, Eap20, Eap30, mic vacuolization and formation of tubulo-vesicular structures and Eap45, which are mammalian homologues of yeast ES- and that overexpression of Alix-CT affected the distribution of CRT-II components, Vps25, Vps22, and Vps36, respectively, an endoplasmic reticulum marker protein, Grp78, but not the are the subunits of RNA polymerase elongation factor, ELL (28, distributions of endosomal marker proteins (EEA1 and Lamp- 51). Thus, some mammalian class E Vps homologues might 1). Taken together with the results of our present study, Alix commonly play dual roles in the regulation of the gene expres- seems to have at least two different functions: a role in protein sion in the nucleus and in the regulation of the MVB sorting in sorting of ubiquitinated endosomal cargoes and an unknown the cytoplasm. function involving the morphology of the endoplasmic reticu- Exosome is a specific form of the MVB inner vesicles secreted lum. The former and the latter functions may be governed by from various cells (52). The ´ ry et al. (11) have revealed that Alix the N-terminal and the C-terminal halves of Alix, respectively. is present in exosomes. This fact agrees with our proposal that We have shown that CHMP4b interacts with Alix by co- Alix is involved in the MVB sorting pathway. Alix may be immunoprecipitation experiments (Figs. 2B and 3) as well as recruited to the inner vesicles of MVB. Future studies, includ- by pull-down assays (Fig. 2A). In these experiments, we ob- ing electron microscopic analysis and tracing of endogenous served that FLAG-CHMP4b proteins from FLAG-CHMP4b/ proteins of Alix and CHMP4b with specific antibodies during HEK293 cells recovered in the soluble fraction in lysis buffer cell surface receptor down-regulation, should clarify the spe- containing 0.2% Nonidet P-40, but when an AlixC-V5-ex- cific stage of endosomal sorting regulated by these mammalian pressing vector was transfected to FLAG-CHMP4b/HEK293 Vps protein homologues. cells, approximately half of the FLAG-CHMP4b proteins were Acknowledgment—We thank Dr. K. Hitomi for valuable discussion. recovered in the insoluble fraction using the same buffer. In contrast, we observed little change in the recovery of FLAG- Note Added in Proof—While a revised version of the manuscript was CHMP4b in the soluble fraction in the case of using transfec- in preparation, the Aspergillus nidulans Vps32 homologue was re- tants of Alix-V5 (data not shown). This fact may be explained ported to interact with human Alix as well as with the fungal homo- logue PalA by the yeast two-hybrid analysis (Vincent, O., Rainbow, L., by the differences in the percentages of cells that formed vesi- Tilburn, J., Arst, H. N., Jr., and Penalva, M. A. (2003) Mol. Cell. Biol. cle-like structures induced by overexpression of Alix-V5 or 23, 1647–1655). Alix and PalA were also shown to recognize a YPXL/I AlixC-V5 in FLAG-CHMP4b/HEK293 cells (14% versus 94%). motif in the fungal zinc finger transcription factor PacC, but this motif CHMP4b-Alix complexes located in the vesicle-like structures are probably insoluble in lysis buffer containing 0.2% Nonidet H. Shibata, K. Yamada, H. Takahashi, T. Mizuno, C. Yorikawa, and P-40. Since the expression levels of AlixC-V5 and Alix-V5 M. Maki, manuscript in preparation. were not significantly different (data not shown), other mole- H. Shibata, T. Mizuno, H. Takahashi, and M. 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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

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 40, Issue of October 3, pp. 39104 –39113, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting* Received for publication, February 14, 2003, and in revised form, July 14, 2003 Published, JBC Papers in Press, July 14, 2003, DOI 10.1074/jbc.M301604200 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; also named AIP1) was Alix (ALG-2-interacting protein X) is a 95-kDa protein 2 2 that interacts with an EF-hand type Ca -binding pro- found as a protein that interacts with the Ca -binding protein tein, ALG-2 (apoptosis-linked gene 2), through its C-ter- ALG-2 (apoptosis-linked gene 2) (1) by the yeast two-hybrid minal proline-rich region. In this study, we searched for method using mouse cDNA libraries by two independent proteins that interact with human AlixC (a truncated groups (2, 3). The interaction between Alix and ALG-2 was form not containing the C-terminal region) by using a shown to be Ca -dependent. Human Alix is a 95-kDa protein yeast two-hybrid screen, and we identified two similar that consists of 868 amino acids. It possesses no obvious struc- human proteins, CHMP4a and CHMP4b (chromatin- tural motifs except for coiled-coil regions and a long proline- modifying protein; charged multivesicular body pro- rich region on the C-terminal side. ALG-2 is one of the penta- tein), as novel binding partners of Alix. The interaction EF-hand Ca -binding proteins (4, 5) and interacts with the of Alix with CHMP4b was confirmed by a glutathione N-terminal domains of annexins VII and XI, which show some S-transferase pull-down assay and by co-immunopre- similarities with the proline-rich region of Alix (6, 7). ALG-2 cipitation experiments. Fluorescence microscopic anal- forms a homodimer as well as a heterodimer with another ysis revealed that CHMP4b transiently expressed in penta-EF-hand protein named peflin (8, 9). In a subcellular HeLa cells mainly exhibited a punctate distribution in fractionation experiment, most of Alix was recovered in the the perinuclear area and co-localized with co-expressed cytosolic fraction of fibroblast cells (3). Results of immunofluo- Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker pro- rescent microscopic analysis showed that overexpressed Alix teins, EEA1 (early endosome antigen 1) and Lamp-1 (ly- was present in the cytoplasm but was concentrated in the sosomal membrane protein-1), respectively. Transient perinuclear region and also localized at the cell periphery in overexpression of CHMP4b induced the accumulation of lamellipodia and filopodia (10). Moreover, proteomic analyses ubiquitinated proteins as punctate patterns that were have revealed that Alix is also present in extracellular mi- partly overlapped with the distribution of CHMP4b and crovesicles called exosomes, which are derived from dendritic inhibited the disappearance of endocytosed epidermal cells (11), and phagosomes isolated from macrophage-like growth factor. In contrast, stably expressed CHMP4b in cells (12). HEK293 cells was observed diffusely in the cytoplasm. Homologues of Alix have been found in many organisms. The Transient overexpression of AlixC in stably CHMP4b- Xenopus homologue of Alix, Xp95, was identified as a protein of expressing cells, however, induced formation of vesicle- 95 kDa that was phosphorylated from the first to second mei- like structures in which CHMP4b and AlixC were co- E235Q otic divisions during progesterone-induced oocyte maturation localized. SKD1 , a dominant negative form of the (13). Overexpression of Hp95, a human homologue of Xp95 AAA type ATPase SKD1 that plays critical roles in the identical to human Alix, induced G arrest, promoted detach- endocytic pathway, was co-immunoprecipitated with 1 ment-induced apoptosis (anoikis), and reduced tumorigenicity CHMP4b. Furthermore, CHMP4b co-localized with E235Q SKD1 as punctate patterns in the perinuclear area, in HeLa cells (14, 15). PalA, an Aspergillus nidulans Alix and Alix was induced to exhibit dot-like distributions homologue, is required for alkaline adaptation of the filamen- E235Q overlapped with SKD1 in HeLa cells. These results tous fungi whose pH regulation of gene expression is mediated suggest that CHMP4b and Alix participate in formation by the zinc finger transcription factor PacC (16, 17). Budding of multivesicular bodies by cooperating with SKD1. yeast has two Alix homologues, Rim20p (18) and Bro1 (19). Genetic analysis of Rim20p has revealed that Rim20p is essen- tial for processing of the zinc finger protein Rim101p, a yeast * This work was supported by a Grant-in-Aid for Scientific Research homologue of PacC (18). Bro1 mutations result in a tempera- 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 ture-sensitive osmoremedial growth defect (19). Although their of page charges. This article must therefore be hereby marked “adver- functions are not known, Alix homologues have also been found tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate in Caenorhabditis elegans (named YNK1 (20)) and in Arabi- this fact. TM The nucleotide sequence(s) reported in this paper has been submitted dopsis (GenBank accession number AC007591, protein TM to the GenBank /EBI Data Bank with accession number(s) AB100261 ID AAD3964201). and AB100262 (at the early stage of this study, we designated CHMP4a In addition to ALG-2, some binding partners of Alix have and CHMP4b as Shax2 and Shax1, respectively). been identified. They are the glioma-associated protein SETA To whom correspondence should be addressed. Tel.: 81-52-789-4088; Fax: 81-52-789-5542; E-mail: [email protected]. (21), which is one of the isoforms of CIN85/Ruk (22, 23), and 39104 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. CHMP4b as Alix-interacting Protein 39105 cDNA fragments obtained by digestion with restriction enzymes were endophilins (10). These proteins, including ALG-2, associate inserted into pEGFP-C2 or pEGFP-C3 (Clontech) vectors. To delete the with the proline-rich region in Alix by Src homology 3 domains putative 5-untranslated region of CHMP4b by the PCR method, the in the case of SETA and endophilins or by a penta-EF-hand forward primer containing a translational initiation codon (underlined), domain in the case of ALG-2. Conservation among the homo- 5-GCGAATTCCATGTCGGTGTTCGGGAAG-3, and the reverse logues, however, is lower within their C-terminal proline-rich primer containing the stop codon (underlined), 5-GCCTCGAGCCATT- regions, and yeast Rim20p lacks this region. In contrast, the ACATGGATCCAGCCCA-3, were designed on the basis of the CHMP4b cDNA sequence. The PCR product was cloned into a pKF3 (TAKARA N-terminal parts of Alix homologues show strong similarities, Shuzo, Kyoto, Japan). To create a pCMV-3FLAG vector for eukaryotic particularly in the regions containing a consensus site of po- expression, forward oligonucleotides, 5-GATCGACTACAAAGACCAT- tential tyrosine phosphorylation by Src family tyrosine ki- GACATCGATTATAAGGATGACGACGAT-3 and 5-GGGATCCCTGC- nases. We speculated that the N-terminal region of Alix plays A-3, and reverse oligonucleotides, 5-TCATCCTTATAATCGATGTCA- an important role in functions common to all Alix homologues. TGGTCTTTGTAGTC-3 and 5-GGGATCCCATCG-3, were inserted In this study, we screened for proteins binding to the N- between BamHI and PstI sites of the pCMV-Tag2C vector (Stratagene). A fragment of the full-length CHMP4b cDNA was inserted into a terminal region of human Alix by the yeast two-hybrid method, pCMV-3FLAG vector and a pEGFP-N1 vector (Clontech), and the and we isolated two clones encoding proteins that are similar to resultant plasmids, designated pFLAG-CHMP4b and pCHMP4b-GFP, each other. One is identical to HSPC134/CHMP4 (chromatin- were used to express 3FLAG-tagged protein and GFP fusion protein, modifying protein; charged multivesicular body protein) (24, respectively. The plasmid encoding GFP-LC3 protein was constructed E235Q 25), which is designated as CHMP4a in this paper, and the as described previously (30). A Myc-SKD1 fragment from pEGFP- EQ other is a novel protein designated as CHMP4b. These CHMP4 MSKD1 (31) was inserted into the EcoRI site of a pCI-neo vector E235Q (Promega) to generate pMyc-SKD1 . proteins are highly homologous to yeast Snf7/Vps32 (vacuolar Yeast Two-hybrid Screening—The MATCHMAKER two-hybrid sys- protein sorting) (26, 27). Yeast Snf7/Vps32 is a member of class tem, including yeast strains and vectors, was obtained from Clontech, E Vps proteins, which are required for sorting of membrane and the screening was performed as described previously (6). Positive proteins into inner vesicles that originate by inward invagina- clones were sequenced using an automated fluorescent sequencer, ABI tion in the lumen of the late endosome (multivesicular body PRISM 310 (PE Applied Biosystems). A computer homology search (MVB) ) (28). Subcellular localization of CHMP4b and the ef- using the advanced BLAST program and coiled-coil region prediction were performed on the World Wide Web. Phylogenetic analysis using fect of CHMP4b overexpression were analyzed, and the ob- Clustal X was performed as described previously (32). tained results suggest that Alix and CHMP4b participate in the Cell Culture and Transfection—HEK293 and HeLa cells were cul- endosomal sorting pathway. tured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), and EXPERIMENTAL PROCEDURES streptomycin (100 g/ml) at 37 °C under humidified air containing 5% Antibodies—Mouse monoclonal antibodies were purchased: EEA1 CO . One day after HEK293 cells or HeLa cells had been seeded, the (early endosome antigen 1; Transduction Laboratories), Lamp-1 (lyso- cells were transfected with the expression plasmid DNAs by the con- somal membrane protein-1)/CD107a (Pharmingen), multiubiquitin ventional calcium phosphate precipitation method or by using Fu- (FK2) (MBL), green fluorescent protein (GFP) (Santa Cruz Biotechnol- GENE6 (Roche Applied Science). After 24 h, cells were harvested and ogy, Inc., Santa Cruz, CA), FLAG tag (M2), c-Myc (9E10) (Sigma), and analyzed. To generate HEK293 stable transfectants that constitutively V5 tag (Invitrogen). Rabbit polyclonal antibodies against FLAG tag and express 3FLAG-tagged CHMP4b (FLAG-CHMP4b/HEK293), GFP were purchased from Sigma and Abcam, respectively. Anti-cation- HEK293 cells transfected with the expression vector (pFLAG-CHMP4b) independent mannose 6-phosphate receptor (anti-CI-MPR) antibody were selected by culturing in the presence of 0.5 mg/ml of G418 for was prepared as described previously (29). Peroxidase-conjugated goat 2 weeks. anti-rabbit and anti-mouse IgG antibodies were from Wako (Osaka, Expression and Purification of GST-AlixC—Escherichia coli BL21 Japan). Cy3-labeled anti-rabbit and anti-mouse antibodies and fluores- cells were transformed with the plasmid pGST-AlixC. The GST fusion cein isothiocyanate-conjugated anti-mouse antibodies used for indirect protein was purified by binding to glutathione-Sepharose 4B (Amer- immunofluorescence analyses were purchased from Amersham Bio- sham Biosciences) according to the manufacturer’s instructions. GST- sciences and Rockland, respectively. AlixC was eluted from Sepharose beads with 10 mM reduced glutathi- Plasmid Constructions—Human Alix cDNA was cloned from Human one in 50 mM Tris-HCl, pH 8.5, dialyzed against 50 mM Tris-HCl, pH TM Fetus Marathon-Ready cDNA (Clontech) by the PCR method and 7.5, and then stored at 4 °C until use. inserted into a pCR2.1-TOPO vector (Invitrogen). pCR2.1-TOPO-Alix Pull-down Assay—One day after FLAG-CHMP4b/HEK293 (6  10 ) Y319F, which has a point mutation at amino acid 319 (from tyrosine to cells had been seeded, the cells were harvested and washed with PBS phenylalanine), was created by PCR-based site-directed mutagenesis (137 mM NaCl, 2.7 mM KCl, 8 mM Na HPO , 1.5 mM KH PO , pH 7.3) 2 4 2 4 according to the provided instruction for QuickChange site-directed and then lysed in 450 l of lysis buffer (10 mM HEPES-NaOH, pH 7.4, mutagenesis kit from Stratagene using two complementary primers 142.5 mM KCl, 0.2% Nonidet P-40, 0.1 mM pefabloc, 25 g/ml leupeptin, (5-AATGACTTCATTTTTCATGATCGAGTT-3 and 5-AACTCGAT- 1 M E-64, and 1 M pepstatin). Supernatants (each 200 l, obtained by CATGAAAAATGAAGTCATT-3), and the mutation was confirmed by centrifugation at 10,000  g) were incubated with 10 g of GST or DNA sequencing. To obtain the cDNA fragment encoding amino acids GST-AlixC protein that had been immobilized on glutathione-Sepha- 1– 660 (AlixC), the palindromic universal translational terminator, rose for3hat4 °C. After complexes had been washed three times with 5-GCTTAATTAATTAAGC-3, was inserted into the blunt-ended NdeI the lysis buffer, they were subjected to Western blotting analysis using site of the Alix cDNA. A cDNA fragment corresponding to AlixC was anti-FLAG monoclonal antibody (mAb). Signals were detected by either inserted into the yeast expression vector pGBKT7 (Clontech), the glu- the chemiluminescent method using Super Signal West Pico Chemilu- tathione S-transferase (GST) fusion vector pGEX-4T-2 (Amersham Bio- minescent Substrate (Pierce) or diaminobenzidine staining. sciences), and a mammalian expression vector pcDNA6/V5-His (Invitro- Immunoprecipitation—FLAG-CHMP4b/HEK293 cells transfected gen) to construct pGBKT7-AlixC, pGST-AlixC, and pAlixC-V5, with the expression vector of either AlixC-V5, GFP-Alix mutants, or respectively. Using the SmaI site located upstream of the stop codon in Myc-SKD1 were lysed in the lysis buffer and then subjected to centrif- the Alix cDNA, a cDNA fragment corresponding to amino acid residues ugation at 10,000  g. The supernatants were incubated with anti-V5 1– 847 was inserted into pcDNA6/V5-His to construct pAlix-V5. To mAb, anti-GFP pAb, or anti-FLAG mAb for 1 h and then incubated for generate truncated forms of GFP-Alix fusion proteins, various Alix 1 h after the addition of Protein G-Sepharose 4 Fast Flow (Amersham Biosciences). The Sepharose beads were then washed with the lysis buffer three times and subjected to Western blotting analysis using 1 either anti-V5 mAb, anti-FLAG mAb, anti-GFP mAb, or anti-Myc mAb. The abbreviations used are: MVB, multivesicular body; CI-MPR, Immunofluorescent Staining—HeLa or HEK293 cells grown on cov- cation-independent mannose 6-phosphate receptor; EGF, epidermal erslips were fixed in 4% paraformaldehyde/PBS and permeabilized in growth factor; ESCRT, endosomal sorting complex required for trans- 0.1% Triton X-100/PBS. After blocking with 0.1% gelatin/PBS, the cells port; GFP, green fluorescent protein; GST, glutathione S-transferase; were incubated with primary antibodies either at 4 °C overnight or at HEK, human embryonic kidney; LC3, microtubule-associated protein 1 room temperature for 1 h and then with secondary antibodies at room light chain 3; mAb, monoclonal antibody; PBS, phosphate-buffered sa- line; pAb, polyclonal antibody; E3, ubiquitin-protein isopeptide ligase. temperature for 1 h. Finally, they were mounted with antifading solu- 39106 CHMP4b as Alix-interacting Protein tion (25 mM Tris-HCl (pH 8.7), 10% polyvinyl alcohol, 5% glycerol, 2.5% region of Alix was essential but not sufficient for CHMP4b 1,4-diazobicyclo-(2,2,2)-octane). Immunofluorescences were analyzed interaction. Since the Tyr of Alix is highly conserved among under a confocal laser-scanning microscope, LSM5 PASCAL (Zeiss). Alix homologues in various organisms and serves as a potential Epidermal Growth Factor (EGF) Uptake Assay—The transfected phosphorylation site for Src-type tyrosine kinases, we replaced HeLa cells were incubated with Dulbecco’s modified Eagle’s medium for Tyr of AlixC with phenylalanine to examine whether this 1hat37 °C and then with 0.5 g/ml tetramethylrhodamine-EGF (Mo- lecular Probes, Inc., Eugene, OR) in 0.5 mg/ml bovine serum albumin/ putative phosphorylation site is needed for the interaction. The Dulbecco’s modified Eagle’s medium for 1 h at 4 °C. The cells were interaction between CHMP4b and Y319FC was still detecta- washed and incubated in 10% fetal bovine serum/Dulbecco’s modified ble with a slight decrease in signal. Eagle’s medium for 30 min or6hat37 °C. Fluorescence Microscopic Analysis of Overexpressed CHMP4b—We investigated the subcellular localization of RESULTS CHMP4b using HeLa cells transfected with pFLAG-CHMP4b. Yeast Two-hybrid Screening—To search for Alix-interacting Double immunofluorescent staining was performed using an proteins, the N-terminal region (amino acids 1– 660) of human anti-FLAG antibody (Fig. 4, A, E, I, and M) and either an Alix (AlixC) that was fused to the Gal4 DNA binding domain antibody of protein that is a marker of early endosome (EEA1) was used as bait, and a HeLa cell cDNA library was used as (Fig. 4B), late endosome and lysosome (Lamp-1) (Fig. 4F), or prey in a yeast two-hybrid screen system. Sixty-two positive the trans-Golgi network and late endosome (CI-MPR) (Fig. 4J). clones were isolated from 6  10 transformants. A search of We also used GFP-LC3 as an autophagosomal membrane DNA data bases revealed that the positive clones corresponded marker instead of staining with an antibody (Fig. 4N). We to 12 different proteins. Two of them were presumed to be found that FLAG-CHMP4b was distributed in a punctate man- human homologues of yeast Snf7/Vps32 (26, 27) (Fig. 1), and ner mainly in the perinuclear area (Fig. 4, A, E, I, and M) and they were also similar to each other (60.7% identity; 136 of 224 slightly diffused in the cytoplasm. However, only the diffuse amino acid residues). One was identical to HSPC134 (24), pattern was observed in some cells (data not shown). As shown which was referred to as CHMP4 by Howard et al. (25), and the in merged images of the same panels (Fig. 4, C, G, and K) and other was very similar to a hypothetical protein registered in in images of higher magnification (Fig. 4, D, H, and L), the TM the sequence data bases (DDBJ/GenBank /EMBL accession distribution of FLAG-CHMP4b partly overlapped in the pe- number AL050349). In this paper, we designated HSPC134/ rinuclear region with the distributions of EEA1 and Lamp-1, CHMP4 as CHMP4a and the other as CHMP4b, respectively. respectively, in the pFLAG-CHMP4b-transfected cell (Fig. 4, C, Both CHMP4a and CHMP4b were predicted to contain coiled- left cell, and G, bottom right cell), and the degree of overlapping coil regions (Fig. 1A). Their N-terminal regions are rich in basic was greater than that of CI-MPR (Fig. 4K, center cell). On the residues, whereas their C-terminal regions are acidic. The other hand, GFP-LC3 showed both punctate and diffuse pat- CHMP4 proteins are part of a large CHMP family (25). The terns (Fig. 4N, inset), and its fluorescent signals were also data base search revealed an additional CHMP4 homologue in detected in the nucleus under the present conditions, in which this subfamily in the human genome (Fig. 1B). autophagy was not enhanced by subjecting the cells to nutrient GST Pull-down Assays of AlixC and CHMP4b—We per- starvation. The distribution of GFP-LC3 was not noticeably formed a GST pull-down assay to determine whether the changed by overexpression of FLAG-CHMP4b (Fig. 4N) and did CHMP4b protein from mammalian cells has the capacity to not overlap with that of FLAG-CHMP4b in cells expressing bind to Alix. First, we constructed several plasmids for express- both GFP-LC3 and FLAG-CHMP4b (Fig. 4, O and P). ing various fusion proteins and generated HEK293 cell lines To determine whether the distribution of ubiquitinated pro- constitutively expressing FLAG-CHMP4b (FLAG-CHMP4b/ teins was affected by CHMP4b overexpression, we examined HEK293). After incubation of the lysate of FLAG-CHMP4b/ the localization of ubiquitinated proteins by staining HeLa HEK293 with the GST fusion protein of AlixC (GST-AlixC) cells transfected with pCHMP4b-GFP using anti-ubiquitin that had been immobilized on glutathione-Sepharose beads, mAb (FK2) that recognizes mono- and polyubiquitinated pro- the proteins bound to the beads were subjected to Western teins but not free monoubiquitin (33, 34). In most of the trans- blotting using an anti-FLAG mAb as a probe. Whereas fected cells, the distribution of CHMP4b-GFP was essentially CHMP4b did not bind to the negative control GST beads, it similar to that of FLAG-CHMP4b (Figs. 4 and 5A). As shown in bound to the GST-AlixC beads (Fig. 2A). Fig. 5B, fluorescent signals of ubiquitinated proteins became Co-immunoprecipitation of CHMP4b and AlixC—When the stronger in cells expressing CHMP4b-GFP, particularly in the lysate of FLAG-CHMP4b/HEK293 cells transfected with the perinuclear area, and showed punctate patterns. Furthermore, expression vector of V5-tagged AlixC (pAlixC-V5) was im- ubiquitinated proteins in this area were partially co-localized munoprecipitated with anti-FLAG mAb, the precipitates con- with CHMP4b-GFP (Fig. 5, C and D). When exogenously ex- tained AlixC-V5 as revealed by Western blotting using an- pressed FLAG-tagged ubiquitin was monitored, accumulation ti-V5 mAb (Fig. 2B). Moreover, in a reciprocal experiment, and partial co-localization of FLAG-tagged ubiquitin with GFP- FLAG-CHMP4b was also co-precipitated with AlixC-V5 using tagged CHMP4b were similarly observed (data not shown). anti-V5 mAb for immunoprecipitation (data not shown). Accumulation of ubiquitinated proteins in the cells overex- Co-immunoprecipitation of CHMP4b with Alix Segments— pressing CHMP4b suggests that overexpression of CHMP4b To narrow the CHMP4b binding region in AlixC, we per- may disturb the endosomal sorting pathway. To provide func- formed co-immunoprecipitation analysis of FLAG-CHMP4b and GFP fusions of full-length or various truncated forms of tional data supporting this speculation, we monitored the fate of endocytosed EGF. HeLa cells transfected with pCHMP4b- Alix. The lysates of FLAG-CHMP4b/HEK293 cells transfected with the expression vectors of GFP-Alix mutants were immu- GFP were incubated with Tetramethylrhodamine-EGF at 4 °C for 1 h, washed, and then allowed to uptake EGF at 37 °C for 30 noprecipitated with anti-GFP pAb, and the precipitates were subjected to Western blotting. As shown in Fig. 3, A and B, and min or 6 h. Internalized EGF was observed at 30 min, and summarized in Fig. 3C, we observed interaction of FLAG- noticeable change was not detected between the CHMP4b-GFP CHMP4b with Alix mutants 1– 868, 1– 660, and 1– 423 and expressing cells and the untransfected cells (Fig. 5, E–G). At weaker interaction with Alix mutant 115– 660. No interaction 6 h, fluorescent signals of tetramethylrhodamine-EGF were was detected with Alix mutants 1–329, 221– 660, and 329 – 660. undetectable in most of the untransfected cells, whereas EGF Bro1-rhophilin conserved domain located in the N-terminal remained in the perinuclear region of the cells expressing CHMP4b as Alix-interacting Protein 39107 FIG.1. Primary structure comparison of CHMP4 proteins with Snf7 and other CHMP family members. A, amino acid sequence alignment of CHMP4 proteins and Snf7. Identical residues between CHMP4 and Snf7 are stippled. The amino acid sequence of Snf7 is from TM GenBank /EMBL/DDBJ (Saccharomyces cerevisiae Snf7; P39929). The coiled-coil regions predicted by the COILS program are indicated with bars above the sequence of CHMP4b. B, phylogenetic comparison of CHMP4 subfamily. Using the default setting of Clustal X 1.81, the amino acid sequences were aligned, and a bootstrap tree file was created. The phylogenetic tree was drawn with NJplot. Horizontal branch lengths are drawn TM to scale with the bar indicating 0.1 amino acid replacement/site. A hypothetical protein is indicated by GenBank /EMBL/DDBJ accession number. Chromosomal loci in the human genome are indicated. h, human; Sc, S. cerevisiae. CHMP4b-GFP (Fig. 5, H–J). However, there was a variation in tially concentrated in the perinuclear region of the Alix-V5 the amount of remaining EGF among the CHMP4b-GFP trans- transfectants (Fig. 6B). In contrast, in the co-transfected cells, fectants (data not shown). Alix-V5 was distributed in the perinuclear region (Fig. 6E), and Change in Alix Distribution by Overexpression of most of the Alix was co-localized with CHMP4b (Fig. 6, D and CHMP4b—To determine whether CHMP4b affects the subcel- F). A similar effect was observed using the pAlixC-V5 con- lular localization of Alix, we performed immunostaining of struct (data not shown). HeLa cells transfected with pAlix-V5 (which encodes V5-tagged Formation of Vesicle-like Structures—As shown in Fig. 7A, Alix) alone or both pAlix-V5 and pFLAG-CHMP4b (Fig. 6). unlike obvious punctate patterns of localization of FLAG- Alix-V5 was diffusely distributed in the cytoplasm and par- CHMP4b in transiently overexpressed HeLa cells (Fig. 4) and 39108 CHMP4b as Alix-interacting Protein E235Q compartment in the cells expressing SKD1 , a dominant negative form of SKD1. To examine whether CHMP4b also interacts with SKD1 and accumulates in the E235Q compart- ment, we performed immunoprecipitation experiments. The lysate of FLAG-CHMP4b/HEK293 cells transfected with the E235Q expression vectors of either Myc-SKD1 or Myc-SKD1 was immunoprecipitated with anti-FLAG mAb, and the precipi- tates were subjected to Western blotting. As shown in Fig. 8A, whereas the interaction of Myc-SKD1 with FLAG-CHMP4b was not detectable under the condition used, the interaction of E235Q Myc-SKD1 with FLAG-CHMP4b was detected. We then performed immunostaining of HeLa cells transfected with ex- E235Q pression vectors of Myc-SKD1 and CHMP4b-GFP. As shown in Fig. 8, B–D, the distribution of CHMP4b-GFP was E235Q very similar to that of Myc-SKD1 , and there were consid- erable overlaps between their distributions. To investigate whether Alix also accumulates in the E235Q compartment, we further performed immunostaining using HeLa cells express- E235Q ing both Myc-SKD1 and GFP-Alix. The accumulation of a fraction of GFP-Alix was observed, and the increased GFP-Alix E235Q signals overlapped those of Myc-SKD1 (Fig. 8, E–G). DISCUSSION By a yeast two-hybrid screen using AlixC (amino acids 1– 660) as bait, we isolated two clones, which are similar to each other, as novel Alix-interacting proteins. By data base analysis, one of them was found to be identical to HSPC134, whose cDNA was isolated as one of the 300 previously unde- fined genes expressed in CD34 hematopoietic stem/progenitor cells (24), and HSPC134 was designated as CHMP4 by Howard et al. (25). In this paper, we designated HSPC134/CHMP4 as CHMP4a and the other similar protein as CHMP4b. These two proteins are homologues of yeast Snf7/Vps32 (Fig. 1), which is one of the class E Vps proteins and is essential for vacuolar FIG.2. Interaction between CHMP4b and AlixC. A, the lysate protein sorting and transport (28). In addition to CHMP4a and of HEK293 cells constitutively expressing FLAG-CHMP4b was incu- CHMP4b, we found another CHMP4 homologue in the human bated with glutathione-Sepharose beads that carried GST or GST- genome data base (Fig. 1B). Their expression patterns in tis- AlixC, and the beads then were pelleted by centrifugation. The pellets were analyzed by Western blotting with anti-FLAG mAb. B, HEK293 sues and during development need to be analyzed in cells constitutively expressing FLAG-CHMP4b were transfected with future studies. pAlixC-V5. After 24 h, transfectants were lysed and immunoprecipi- In general, coiled-coil regions mediate the protein-protein tated (IP) with anti-FLAG mAb (FLAG) or control mouse IgG (con). The interactions. Since both Alix and CHMP4b have coiled-coil immunoprecipitates were analyzed by Western blotting (WB) with an- regions, at first we speculated that these regions were essential ti-V5 mAb or anti-FLAG mAb. Mouse immunoglobulin heavy chains (IgG-H) and light chains (IgG-L) in the immunoprecipitates were also for the interaction between the two proteins. However, as detected by subsequent Western blotting using peroxidase-conjugated shown in Fig. 3, the coiled-coil regions in Alix were not required anti-mouse IgG as a secondary antibody. The molecular masses of for their interaction, and the determined CHMP4b-interacting standard proteins are indicated on the left. region possesses a potential phosphorylation site by Src-type tyrosine kinases. Indeed, substitution of Tyr with Phe in HEK293 cells (data not shown), FLAG-CHMP4b showed (Y319F mutant) has been shown to lose the ability to interact diffuse cytoplasmic distribution in stably expressing cells with both focal adhesion kinase and PYK-2 (35). The Y319F (FLAG-CHMP4b/HEK293), but vesicle-like structures were ob- mutant, however, could substantially interact with CHMP4b served in 5% of the cells (data not shown). On the other hand, (Fig. 3A). Xu et al. (18) reported that Rim20p, which is another Alix-V5 and AlixC-V5 were localized in the cytoplasm, even in yeast homologue of Alix, associates with Rim101p regardless of HEK293 cells transiently transfected with pAlix-V5 (Fig. 7B) substitutions of Ile-Tyr to Ala-Ala in this highly conserved or pAlixC-V5 (Fig. 7C). In the FLAG-CHMP4b/HEK293 cells region. Although Tyr in human Alix is widely conserved transfected with pAlixC-V5, FLAG-CHMP4b and AlixC-V5 from lower to higher eukaryotic cells, the importance of Tyr showed punctate patterns of distribution (Fig. 7, G, H, J, and for protein complex formation appears to be dependent on K) in 94% of the cells examined, and these two proteins were interacting partners. co-localized in vesicle-like structures (Fig. 7, I and L, indicated In yeast cells, class E Vps proteins are essential for carboxyl by the arrowheads). In contrast, the effect of overexpression of peptidase S sorting, and a group of class E Vps proteins form Alix-V5 in FLAG-CHMP4b/HEK293 cells on CHMP4b localiza- three complexes, named ESCRT-I, -II, and -III (endosomal tion was not significant (Fig. 7, D, E, and F), and vesicle-like sorting complex required for transport) (36 –38). ESCRTs work structures stained with FLAG-CHMP4b and Alix-V5 were ob- in the sorting of ubiquitinated proteins in the process of the served in only 14% of the cells examined (data not shown). invaginating endosomal membrane toward the lumen, and this Accumulation of CHMP4b and Alix at the E235Q Compart- compartment containing inner vesicles is called the MVB. ES- ment—Howard et al. (25) reported that CHMP1 interacts with CRT-III is composed of four similar yeast CHMP family pro- SKD1, which is an AAA-type ATPase homologous to yeast teins, among which Snf7 forms a subcomplex with Vps20 and Vps4, and that CHMP1 accumulates in the so-called E235Q Vps2 forms a subcomplex with Vps24. It has recently been CHMP4b as Alix-interacting Protein 39109 FIG.3. Immunoprecipitation of CHMP4b with Alix truncated mutants. A and B, HEK293 cells constitutively expressing FLAG-CHMP4b were transfected with the expression vectors for various GFP-Alix truncated mutants. After 24 h, transfectants were lysed and immunoprecipitated with anti-GFP pAb. The immunoprecipitates were analyzed by Western blotting (WB) with anti-GFP mAb or anti-FLAG mAb. The numbers indicate the N-terminal and C-terminal Alix residues of the truncation mutants. C, a schematic representation of Alix truncated mutants is shown. The numbers indicate the N-terminal and C-terminal Alix residues of mutants. Y, the tyrosine residue in a highly conserved potential recognition site for Src type tyrosine kinases. F, the substituted phenylalanine residue by the point mutation. The relative strengths of the interactions are indicated as follows. , equivalent to full-length; , slightly weak; , weak; , not detectable. BRD, Bro1-rhophilin conserved domain (NCBI conserved domain data base accession number 17278; pfam03097); CC, coiled-coil; PRR, proline-rich region. reported that Bro1 (yeast homologue of Alix) is identical to cells, the amount of the FLAG-CHMP4b protein in the tran- Vps31, which is also a class E Vps protein (28, 39). Further, the siently expressed HEK293 cells was estimated about 1.5–2 endosomal association of Bro1 has been shown to be specifically times as much as the amount of the protein expressed in the dependent on Snf7 (40). Although direct interaction between FLAG-CHMP4b/HEK293 cells (data not shown). Considering Bro1/Vps31 and Snf7/Vps32 has not been demonstrated, the the transfection efficiency of 25–50%, the FLAG-CHMP4b pro- association of these proteins was suggested by results of sys- tein in an individual transient transfectant was calculated to tematic analysis of protein complexes by mass spectrometry be present 3– 8 times more per cell than in the stably express- (41). Moreover, the interaction between another yeast Alix ing cells. Thus, the punctate subcellular localization of exog- homologue, Rim20p, and Snf7 was also suggested by results of enously expressed CHMP4b may depend on the expression genome-wide two-hybrid analysis (42). Since both CHMP4 and levels but not on the types of the cells. Indeed, diffuse cytoplas- Alix are widely present from lower to higher eukaryotic cells, mic distributions were also observed in some HeLa cells by the function mediated by the interaction between the two pro- transient expression of the N-terminally tagged proteins teins is presumed to be conserved and seems to be involved in (FLAG-CHMP4b and GFP-CHMP4b), particularly, using the regulation of sorting in the endosomal pathway. lesser amounts of expression vectors (data not shown). Since In HeLa cells (Figs. 4 and 5) and also in HEK293 cells (data the C-terminally tagged CHMP4b-GFP protein is more prone not shown), most of the transiently overexpressed CHMP4b to exhibit cytoplasmic punctate distributions under the similar proteins are distributed in punctate patterns mainly in the transfection condition, tagging at the N terminus of CHMP4b perinuclear area. On the other hand, in HEK293 cells stably might affect its stability, folding, and/or interaction with other expressing FLAG-CHMP4b (FLAG-CHMP4b/HEK293), the proteins. diffuse distribution of CHMP4b in the cytoplasm was observed As in the yeast system, where the dissociation of Snf7 from (Fig. 7A). By Western blot analysis using similar numbers of the limiting membrane of MVB has been shown to depend on 39110 CHMP4b as Alix-interacting Protein FIG.6. Effect of FLAG-CHMP4b overexpression on the distri- bution of Alix-V5. HeLa cells transfected with the pAlix-V5 alone (A–C) or both pAlix-V5 and pFLAG-CHMP4b (D--F) were subjected to immunofluorescence confocal microscopy using anti-FLAG pAb (A and D) and anti-V5 mAb (B and E). A and D, FLAG-CHMP4b; B and E, Alix-V5; C and F, merged images of either A and B or D and E. Bar, 10 m. FIG.4. Subcelluar localization of CHMP4b in HeLa cells. HeLa cells transfected with pFLAG-CHMP4b alone (A–L) or both pFLAG- CHMP4b and pEGFP-C1-LC3 (M–P) were subjected to immunofluores- cence confocal microscopy using polyclonal (A, E, and M) or monoclonal (I) anti-FLAG antibodies and anti-endosomal marker protein antibod- ies (anti-EEA1 mAb, anti-Lamp1 mAb, or anti-CI-MPR pAb). A, E, I, and M, immunofluorescent micrographs of FLAG-CHMP4b; B, EEA1; F, Lamp-1; J, CI-MPR; N, fluorescent micrographs of GFP-LC3; C, G, K, and O, merged images; D, H, L, and P, higher magnifications of images shown in C, G, K, and O; insets in M and N, the cells expressing GFP-LC3 alone in a different field. Bar,10 m. FIG.7. Formation of vesicle-like structures and CHMP4b ac- cumulation by AlixC overexpression. HEK293 cells (B and C)or FIG.5. Effects of CHMP4b overexpression on the endocytic HEK293 cells constitutively expressing FLAG-CHMP4b (FLAG- pathway. A–D, HeLa cells transfected with pCHMP4b-GFP were CHMP4b/HEK293) (A and D–L) transfected with pAlix-V5 (B and D–F) stained with anti-ubiquitin mAb (FK2). A, CHMP4b-GFP; B, ubiquitin; or pAlixC-V5 (C and G–L) were subjected to immunofluorescence C, merged image of A and B; D, higher magnification of the image confocal microscopy using anti-FLAG pAb (A, D, and G) and anti-V5 shown in C. E–J, HeLa cells transfected with CHMP4b-GFP were mAb (B, C, E, and H). F and I, merged images of either G and H or J and incubated at 4 °Cfor1hinthe presence of tetramethylrhodamine-EGF, K. J–L, higher magnification of images shown in G–I, respectively. The washed, and then incubated at 37 °C for 30 min (E–G)or6h(H–J). E arrowheads indicate the vesicle-like structures. Bar,10 m. and H, CHMP4b-GFP; F and I, tetramethylrhodamine-EGF; G and J, merged images. The asterisks indicate untransfected cells. Bars in C, G, and J,10 m; D,2 m. E235Q tion. CHMP4b was co-immunoprecipitated with SKD1 , the ATPase activity of Vps4 (27), the localization of CHMP4b which is a SKD1 mutant defective in ATP hydrolysis, but not may be also regulated by SKD1, a mammalian homologue of with wild type SKD1 (Fig. 8A). This result agrees with the Vps4 (43, 44). In the present study, this hypothesis was exam- previous report that blocking ATP hydrolysis of an AAA type ined first by analysis of the interaction between CHMP4b and ATPase by mutation (Glu to Gln mutation in the Walker B box) SKD1 and then by analysis of their intracellular co-localiza- results in high affinity interactions of the AAA protein with its CHMP4b as Alix-interacting Protein 39111 E235Q E235Q FIG.8. Interaction between CHMP4b and SKD1 and co-localization of CHMP4b and Alix with SKD1 . A, HEK293 cells E235Q constitutively expressing FLAG-CHMP4b were transfected with either pMyc-SKD1 or pMyc-SKD1 . After 24 h, transfectants were lysed and immunoprecipitated (IP) with anti-FLAG mAb (FLAG) or control mouse IgG (con). The immunoprecipitates were analyzed by Western blotting (WB) with anti-FLAG mAb or anti-Myc mAb. IgG-H and IgG-L indicate mouse immunoglobulin heavy chains and light chains, respectively. B–G, E235Q HeLa cells co-transfected with the expression vectors of Myc-SKD1 and either CHMP4b-GFP (B–D) or GFP-Alix (E–G) were subjected to E235Q immunofluorescence confocal microscopy using anti-Myc mAb. B, CHMP4b-GFP; E, GFP-Alix; C and F, Myc-SKD1 ; D and G, merged images of either B and C or E and F. Bar,10 m. protein targets (45). We observed the co-localization of CHMP4b was partially co-localized with EEA1 and Lamp-1 E235Q CHMP4b with SKD1 in HeLa cells expressing CHMP4b- and to a lesser degree with CI-MPR (a marker protein for late E235Q GFP and Myc-SKD1 (Fig. 8, B–D). Furthermore, the co- endosome and trans-Golgi network), suggesting localization of localization of CHMP4b-GFP with endogenous SKD1 was ob- CHMP4b from early to late endosomes. Ubiquitinated proteins served using anti-SKD1 antibody (data not shown). Since accumulated in cells overexpressing CHMP4b, and CHMP4b excessive CHMP4b in the transiently expressed cells may se- was partly co-localized with ubiquitinated proteins like other quester endogenous SKD1, CHMP4b molecules may not be able endosomal marker proteins (Fig. 5, A–D). The electrophoretic to dissociate from the endosomal membranes and may keep mobilities of the FLAG-CHMP4b proteins analyzed by Western associating with other ESCRT components. The accumulation blotting using anti-FLAG antibody were not different between of Alix in the E235Q compartment was observed as in the case the lysates prepared from diffusely stained cells (FLAG- of CHMP4b (Fig. 8, E–G). This result suggests that the disso- CHMP4b/HEK293) and those from transiently overexpressed ciation of Alix from endosomes might also depend on the HeLa cells that exhibited punctate distributions, and no higher ATPase activity of SKD1. molecular mass FLAG-CHMP4b proteins indicative of ubiquiti- The distributions of endosomal marker proteins such as nation were observed in either cell lysates by Western blotting EEA1 (early endosome) and Lamp-1 (late endosome and lyso- (data not shown). Thus, the observed co-localization of the some) were affected by overexpression of CHMP4b (Fig. 4). ubiquitinated proteins and CHMP4b-GFP shown in Fig. 5, 39112 CHMP4b as Alix-interacting Protein A–D, may not be due to ubiquitination of the CHMP4 protein have an important role for dissociation from endosomal mem- itself. Ubiquitination of proteins is not only a signal of protea- branes. Our recently obtained data indicate that the binding somal degradation of cellular proteins but also a triggering sites for ALG-2, CIN85, and endophilin are all located at three signal for sorting of plasma membrane proteins such as growth different sites within the C-terminal proline-rich region. Therefore, association or dissociation of Alix with these factors factor receptors to lysosomal degradation through the endoso- mal sorting pathway, where Hrs and TSG101 play roles in may trigger a conformational change of Alix and may affect the ability of Alix to interact with CHMP4b. Interestingly, CIN85 directing ubiquitinated proteins via their ubiquitin-binding do- and endophilin are also factors involved in membrane receptor mains to the components of MVB sorting on endosomal mem- internalization triggered by Cbl, which has intrinsic ubiquiti- branes (28, 46). Thus, the accumulation of the ubiquitinated nation activities as a RING-type E3 ligase (47, 48). proteins (Fig. 5, A–D) and the finding that overexpression of Springael et al. (39) reported that Npi3, which is identical to CHMP4b affected the distribution of endosomal markers (Fig. Bro1, is required for vacuolar sorting of Gap1 permease under 4) suggest disturbance of the endosomal protein sorting path- ubiquitin-dependent control in budding yeast. Compared with way by overexpressed CHMP4b proteins. To provide experi- the genetically well studied yeast system, information on the mental evidence in favor of this speculation, we monitored a mammalian MVB sorting pathway is still limited. Recent data, fate of endocytosed EGF. Disappearance of the fluorescent sig- however, indicate that factors involving ubiquitin actions in the nals of labeled EGF was inhibited in the HeLa cells transfected pathway are conserved between yeast and mammals (e.g. with CHMP4b-GFP (Fig. 5, H–J), raising a possibility that Vps27 versus Hrs, Vps23 versus TSG101, and Rsp5 versus overexpression of CHMP4b disturbs the regulation of mem- Nedd4) (28, 49). Interestingly, TSG101 was isolated as a can- brane trafficking from endosomes to lysosomes. didate cDNA clone of the Alix-interacting partner using a frag- Overexpression of CHMP4b caused accumulation of Alix ment containing the proline-rich region of Alix as bait by the from the cytoplasm to the perinuclear area, where CHMP4b yeast two-hybrid screen. Alix might link components of ubiq- was co-localized (Fig. 6). We also found that overexpression of uitin-dependent internalization machinery to components of AlixC-V5 induced formation of vesicle-like structures in cells the MVB sorting complexes by interacting with multiple stably expressing FLAG-CHMP4b (FLAG-CHMP4b/HEK293) factors. (Fig. 7, G–L). These findings suggest that Alix cooperates with Although the exogenously expressed FLAG-CHMP4b pro- CHMP4b and participates in the endosomal pathway. Since no teins exhibited cytoplasmic punctate patterns, they were also vesicular structures were observed in HEK293 cells transfected observed in the nucleus in some cells (Figs. 4 and 7). CHMP1, with pAlixC-V5 (Fig. 7C), the level of endogenous CHMP4b which is implicated in MVB formation (25), was first found as seems to be too low to induce vesicle formation. In contrast, a partner of Polycomb group protein Pcl (Polycomblike) by a transient overexpression of CHMP4b both in HeLa cells (Figs. yeast two-hybrid screen, and a role in stable gene silencing 4 and 5) and in HEK293 cells (data not shown) induced the within the nucleus was suggested (50). A post-translationally accumulation of CHMP4b in the perinuclear area as punctate modified form of CHMP1 is associated with nuclear matrix, patterns without exogenous Alix, suggesting a sufficient ex- and overexpressed CHMP1 localizes to a punctate subnuclear pression level of endogenous Alix for CHMP4b interaction. pattern and arrests cells in S-phase (50). On the other hand, Previously, Chatellard-Causse et al. (10) reported that overex- some mammalian class E Vps homologues were identified as pression of Alix-CT, the C-terminal half of Alix, led to cytoplas- components of nuclear factors. For instance, Eap20, Eap30, mic vacuolization and formation of tubulo-vesicular structures and Eap45, which are mammalian homologues of yeast ES- and that overexpression of Alix-CT affected the distribution of CRT-II components, Vps25, Vps22, and Vps36, respectively, an endoplasmic reticulum marker protein, Grp78, but not the are the subunits of RNA polymerase elongation factor, ELL (28, distributions of endosomal marker proteins (EEA1 and Lamp- 51). Thus, some mammalian class E Vps homologues might 1). Taken together with the results of our present study, Alix commonly play dual roles in the regulation of the gene expres- seems to have at least two different functions: a role in protein sion in the nucleus and in the regulation of the MVB sorting in sorting of ubiquitinated endosomal cargoes and an unknown the cytoplasm. function involving the morphology of the endoplasmic reticu- Exosome is a specific form of the MVB inner vesicles secreted lum. The former and the latter functions may be governed by from various cells (52). The ´ ry et al. (11) have revealed that Alix the N-terminal and the C-terminal halves of Alix, respectively. is present in exosomes. This fact agrees with our proposal that We have shown that CHMP4b interacts with Alix by co- Alix is involved in the MVB sorting pathway. Alix may be immunoprecipitation experiments (Figs. 2B and 3) as well as recruited to the inner vesicles of MVB. Future studies, includ- by pull-down assays (Fig. 2A). In these experiments, we ob- ing electron microscopic analysis and tracing of endogenous served that FLAG-CHMP4b proteins from FLAG-CHMP4b/ proteins of Alix and CHMP4b with specific antibodies during HEK293 cells recovered in the soluble fraction in lysis buffer cell surface receptor down-regulation, should clarify the spe- containing 0.2% Nonidet P-40, but when an AlixC-V5-ex- cific stage of endosomal sorting regulated by these mammalian pressing vector was transfected to FLAG-CHMP4b/HEK293 Vps protein homologues. cells, approximately half of the FLAG-CHMP4b proteins were Acknowledgment—We thank Dr. K. Hitomi for valuable discussion. recovered in the insoluble fraction using the same buffer. In contrast, we observed little change in the recovery of FLAG- Note Added in Proof—While a revised version of the manuscript was CHMP4b in the soluble fraction in the case of using transfec- in preparation, the Aspergillus nidulans Vps32 homologue was re- tants of Alix-V5 (data not shown). This fact may be explained ported to interact with human Alix as well as with the fungal homo- logue PalA by the yeast two-hybrid analysis (Vincent, O., Rainbow, L., by the differences in the percentages of cells that formed vesi- Tilburn, J., Arst, H. N., Jr., and Penalva, M. A. (2003) Mol. Cell. Biol. cle-like structures induced by overexpression of Alix-V5 or 23, 1647–1655). Alix and PalA were also shown to recognize a YPXL/I AlixC-V5 in FLAG-CHMP4b/HEK293 cells (14% versus 94%). motif in the fungal zinc finger transcription factor PacC, but this motif CHMP4b-Alix complexes located in the vesicle-like structures are probably insoluble in lysis buffer containing 0.2% Nonidet H. Shibata, K. Yamada, H. Takahashi, T. Mizuno, C. Yorikawa, and P-40. Since the expression levels of AlixC-V5 and Alix-V5 M. Maki, manuscript in preparation. were not significantly different (data not shown), other mole- H. Shibata, T. Mizuno, H. Takahashi, and M. 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Published: Sep 26, 2003

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