The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m301604200
ISSN1083-351X
AutoresKeiichi Katoh, Hideki Shibata, Hidenori Suzuki, Atsuki Nara, Kazumi Ishidoh, Eiki Kominami, Tamotsu Yoshimori, Masatoshi Maki,
Tópico(s)Fungal and yeast genetics research
ResumoAlix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2+-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixΔC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixΔC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixΔC were co-localized. SKD1E235Q, a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1E235Q as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1E235Q in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1. Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2+-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixΔC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixΔC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixΔC were co-localized. SKD1E235Q, a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1E235Q as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1E235Q in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1. Alix (ALG-2-interacting protein X; also named AIP1) was found as a protein that interacts with the Ca2+-binding protein ALG-2 (apoptosis-linked gene 2) (1Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar) by the yeast two-hybrid method using mouse cDNA libraries by two independent groups (2Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 3Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The interaction between Alix and ALG-2 was shown to be Ca2+-dependent. Human Alix is a 95-kDa protein that consists of 868 amino acids. It possesses no obvious structural motifs except for coiled-coil regions and a long proline-rich region on the C-terminal side. ALG-2 is one of the penta-EF-hand Ca2+-binding proteins (4Maki M. Yamaguchi K. Kitaura Y. Satoh H. Hitomi K. J. Biochem. (Tokyo). 1998; 124: 1170-1177Crossref PubMed Scopus (53) Google Scholar, 5Maki M. Kitaura Y. Satoh H. Ohkouchi S. Shibata H. Biochim. Biophys. Acta. 2002; 1600: 51-60Crossref PubMed Scopus (156) Google Scholar) and interacts with the N-terminal domains of annexins VII and XI, which show some similarities with the proline-rich region of Alix (6Satoh H. Shibata H. Nakano Y. Kitaura Y. Maki M. Biochem. Biophys. Res. Commun. 2002; 291: 1166-1172Crossref PubMed Scopus (58) Google Scholar, 7Satoh H. Nakano Y. Shibata H. Maki M. Biochim. Biophys. Acta. 2002; 1600: 61-67Crossref PubMed Scopus (63) Google Scholar). ALG-2 forms a homodimer as well as a heterodimer with another penta-EF-hand protein named peflin (8Kitaura Y. Matsumoto S. Satoh H. Hitomi K. Maki M. J. Biol. Chem. 2001; 276: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Kitaura Y. Watanabe M. Satoh H. Kawai T. Hitomi K. Maki M. Biochem. Biophys. Res. Commun. 1999; 263: 68-75Crossref PubMed Scopus (35) Google Scholar). In a subcellular fractionation experiment, most of Alix was recovered in the cytosolic fraction of fibroblast cells (3Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Results of immunofluorescent microscopic analysis showed that overexpressed Alix was present in the cytoplasm but was concentrated in the perinuclear region and also localized at the cell periphery in lamellipodia and filopodia (10Chatellard-Causse C. Blot B. Cristina N. Torch S. Missotten M. Sadoul R. J. Biol. Chem. 2002; 277: 29108-29115Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Moreover, proteomic analyses have revealed that Alix is also present in extracellular microvesicles called exosomes, which are derived from dendritic cells (11Théry C. Boussac M. Veron P. Ricciardi-Castagnoli P. Raposo G. Garin J. Amigorena S. J. Immunol. 2001; 166: 7309-7318Crossref PubMed Scopus (1193) Google Scholar), and phagosomes isolated from macrophage-like cells (12Garin J. Diez R. Kieffer S. Dermine J.F. Duclos S. Gagnon E. Sadoul R. Rondeau C. Desjardins M. J. Cell Biol. 2001; 152: 165-180Crossref PubMed Scopus (609) Google Scholar). Homologues of Alix have been found in many organisms. The Xenopus homologue of Alix, Xp95, was identified as a protein of 95 kDa that was phosphorylated from the first to second meiotic divisions during progesterone-induced oocyte maturation (13Che S. El-Hodiri H.M. Wu C.F. Nelman-Gonzalez M. Weil M.M. Etkin L.D. Clark R.B. Kuang J. J. Biol. Chem. 1999; 274: 5522-5531Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Overexpression of Hp95, a human homologue of Xp95 identical to human Alix, induced G1 arrest, promoted detachment-induced apoptosis (anoikis), and reduced tumorigenicity in HeLa cells (14Wu Y. Pan S. Che S. He G. Nelman-Gonzalez M. Weil M.M. Kuang J. Differentiation. 2001; 67: 139-153Crossref PubMed Scopus (42) Google Scholar, 15Wu Y. Pan S. Luo W. Lin S.H. Kuang J. Oncogene. 2002; 21: 6801-6808Crossref PubMed Scopus (40) Google Scholar). PalA, an Aspergillus nidulans Alix homologue, is required for alkaline adaptation of the filamentous fungi whose pH regulation of gene expression is mediated by the zinc finger transcription factor PacC (16Negrete-Urtusan S. Denison S.H. Arst Jr., H.N. J. Bacteriol. 1997; 179: 1832-1835Crossref PubMed Google Scholar, 17Denison S.H. Fungal Genet. Biol. 2000; 29: 61-71Crossref PubMed Scopus (128) Google Scholar). Budding yeast has two Alix homologues, Rim20p (18Xu W. Mitchell A.P. J. Bacteriol. 2001; 183: 6917-6923Crossref PubMed Scopus (101) Google Scholar) and Bro1 (19Nickas M.E. Yafee M.P. Mol. Cell Biol. 1996; 16: 2585-2593Crossref PubMed Scopus (65) Google Scholar). Genetic analysis of Rim20p has revealed that Rim20p is essential for processing of the zinc finger protein Rim101p, a yeast homologue of PacC (18Xu W. Mitchell A.P. J. Bacteriol. 2001; 183: 6917-6923Crossref PubMed Scopus (101) Google Scholar). Bro1 mutations result in a temperature-sensitive osmoremedial growth defect (19Nickas M.E. Yafee M.P. Mol. Cell Biol. 1996; 16: 2585-2593Crossref PubMed Scopus (65) Google Scholar). Although their functions are not known, Alix homologues have also been found in Caenorhabditis elegans (named YNK1 (20Che S. Weil M.M. Etkin L.D. Epstein H.F. Kuang J. Biochim. Biophys. Acta. 1997; 1354: 231-240Crossref PubMed Scopus (11) Google Scholar)) and in Arabidopsis (GenBank™ accession number AC007591, protein ID AAD3964201). In addition to ALG-2, some binding partners of Alix have been identified. They are the glioma-associated protein SETA (21Chen B. Borinstein S.C. Gillis J. Sykes V.W. Bogler O. J. Biol. Chem. 2000; 275: 19275-19281Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), which is one of the isoforms of CIN85/Ruk (22Take H. Watanabe S. Takeda K. Yu Z.X. Iwata N. Kajigaya S. Biochem. Biophys. Res. Commun. 2000; 268: 321-328Crossref PubMed Scopus (137) Google Scholar, 23Gout I. Middleton G. Adu J. Ninkina N.N. Drobot L.B. Filonenko V. Matsuka G. Davies A.M. Waterfield M. Buchman V.L. EMBO J. 2000; 19: 4015-4025Crossref PubMed Scopus (121) Google Scholar), and endophilins (10Chatellard-Causse C. Blot B. Cristina N. Torch S. Missotten M. Sadoul R. J. Biol. Chem. 2002; 277: 29108-29115Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). These proteins, including ALG-2, associate with the proline-rich region in Alix by Src homology 3 domains in the case of SETA and endophilins or by a penta-EF-hand domain in the case of ALG-2. Conservation among the homo-logues, however, is lower within their C-terminal proline-rich regions, and yeast Rim20p lacks this region. In contrast, the N-terminal parts of Alix homologues show strong similarities, particularly in the regions containing a consensus site of potential tyrosine phosphorylation by Src family tyrosine kinases. We speculated that the N-terminal region of Alix plays an important role in functions common to all Alix homologues. In this study, we screened for proteins binding to the N-terminal region of human Alix by the yeast two-hybrid method, and we isolated two clones encoding proteins that are similar to each other. One is identical to HSPC134/CHMP4 (chromatin-modifying protein; charged multivesicular body protein) (24Zhang Q.-H. Ye M. Wu X.-Y. Ren S.-X. Zhao M. Zhao C.-J. Fu G. Shen Y. Fan H.-Y. Lu G. Zhong M. Xu X.-R. Han Z.-G. Zhang J.-W. Tao J. Huang Q.-H. Zhou J. Hu G.-X. Gu J. Chen S.-J. Chen Z. Genome Res. 2000; 10: 1546-1560Crossref PubMed Scopus (160) Google Scholar, 25Howard T.L. Stauffer D.R. Degnin C.R. Hollenberg S.M. J. Cell Sci. 2001; 114: 2395-2404Crossref PubMed Google Scholar), which is designated as CHMP4a in this paper, and the other is a novel protein designated as CHMP4b. These CHMP4 proteins are highly homologous to yeast Snf7/Vps32 (vacuolar protein sorting) (26Tu J. Vallier L.G. Carlson M. Genetics. 1993; 135: 17-23Crossref PubMed Google Scholar, 27Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar). Yeast Snf7/Vps32 is a member of class E Vps proteins, which are required for sorting of membrane proteins into inner vesicles that originate by inward invagination in the lumen of the late endosome (multivesicular body (MVB) 1The abbreviations used are: MVB, multivesicular body; CI-MPR, cation-independent mannose 6-phosphate receptor; EGF, epidermal growth factor; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; LC3, microtubule-associated protein 1 light chain 3; mAb, monoclonal antibody; PBS, phosphate-buffered saline; pAb, polyclonal antibody; E3, ubiquitin-protein isopeptide ligase.) (28Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar). Subcellular localization of CHMP4b and the effect of CHMP4b overexpression were analyzed, and the obtained results suggest that Alix and CHMP4b participate in the endosomal sorting pathway. Antibodies—Mouse monoclonal antibodies were purchased: EEA1 (early endosome antigen 1; Transduction Laboratories), Lamp-1 (lysosomal membrane protein-1)/CD107a (Pharmingen), multiubiquitin (FK2) (MBL), green fluorescent protein (GFP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), FLAG tag (M2), c-Myc (9E10) (Sigma), and V5 tag (Invitrogen). Rabbit polyclonal antibodies against FLAG tag and GFP were purchased from Sigma and Abcam, respectively. Anti-cation-independent mannose 6-phosphate receptor (anti-CI-MPR) antibody was prepared as described previously (29Muno D. Ishidoh K. Ueno T. Kominami E. Arch. Biochem. Biophys. 1993; 306: 103-110Crossref PubMed Scopus (49) Google Scholar). Peroxidase-conjugated goat anti-rabbit and anti-mouse IgG antibodies were from Wako (Osaka, Japan). Cy3-labeled anti-rabbit and anti-mouse antibodies and fluorescein isothiocyanate-conjugated anti-mouse antibodies used for indirect immunofluorescence analyses were purchased from Amersham Biosciences and Rockland, respectively. Plasmid Constructions—Human Alix cDNA was cloned from Human Fetus Marathon-Ready™ cDNA (Clontech) by the PCR method and inserted into a pCR2.1-TOPO vector (Invitrogen). pCR2.1-TOPO-Alix Y319F, which has a point mutation at amino acid 319 (from tyrosine to phenylalanine), was created by PCR-based site-directed mutagenesis according to the provided instruction for QuickChange site-directed mutagenesis kit from Stratagene using two complementary primers (5′-AATGACTTCATTTTTCATGATCGAGTT-3′ and 5′-AACTCGATCATGAAAAATGAAGTCATT-3′), and the mutation was confirmed by DNA sequencing. To obtain the cDNA fragment encoding amino acids 1-660 (AlixΔC), the palindromic universal translational terminator, 5′-GCTTAATTAATTAAGC-3′, was inserted into the blunt-ended NdeI site of the Alix cDNA. A cDNA fragment corresponding to AlixΔC was inserted into the yeast expression vector pGBKT7 (Clontech), the glutathione S-transferase (GST) fusion vector pGEX-4T-2 (Amersham Biosciences), and a mammalian expression vector pcDNA6/V5-His (Invitrogen) to construct pGBKT7-AlixΔC, pGST-AlixΔC, and pAlixΔC-V5, respectively. Using the SmaI site located upstream of the stop codon in the Alix cDNA, a cDNA fragment corresponding to amino acid residues 1-847 was inserted into pcDNA6/V5-His to construct pAlix-V5. To generate truncated forms of GFP-Alix fusion proteins, various Alix cDNA fragments obtained by digestion with restriction enzymes were inserted into pEGFP-C2 or pEGFP-C3 (Clontech) vectors. To delete the putative 5′-untranslated region of CHMP4b by the PCR method, the forward primer containing a translational initiation codon (underlined), 5′-GCGAATTCCATGTCGGTGTTCGGGAAG-3′, and the reverse primer containing the stop codon (underlined), 5′-GCCTCGAGCCATTACATGGATCCAGCCCA-3′, were designed on the basis of the CHMP4b cDNA sequence. The PCR product was cloned into a pKF3 (TAKARA Shuzo, Kyoto, Japan). To create a pCMV-3×FLAG vector for eukaryotic expression, forward oligonucleotides, 5′-GATCGACTACAAAGACCATGACATCGATTATAAGGATGACGACGAT-3′ and 5′-GGGATCCCTGCA-3′, and reverse oligonucleotides, 5′-TCATCCTTATAATCGATGTCATGGTCTTTGTAGTC-3′ and 5′-GGGATCCCATCG-3′, were inserted between BamHI and PstI sites of the pCMV-Tag2C vector (Stratagene). A fragment of the full-length CHMP4b cDNA was inserted into a pCMV-3×FLAG vector and a pEGFP-N1 vector (Clontech), and the resultant plasmids, designated pFLAG-CHMP4b and pCHMP4b-GFP, were used to express 3×FLAG-tagged protein and GFP fusion protein, respectively. The plasmid encoding GFP-LC3 protein was constructed as described previously (30Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar). A Myc-SKD1E235Q fragment from pEGFPMSKD1EQ (31Nara A. Mizushima N. Yamamoto A. Kabeya Y. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 29-37Crossref PubMed Scopus (123) Google Scholar) was inserted into the EcoRI site of a pCI-neo vector (Promega) to generate pMyc-SKD1E235Q. Yeast Two-hybrid Screening—The MATCHMAKER two-hybrid system, including yeast strains and vectors, was obtained from Clontech, and the screening was performed as described previously (6Satoh H. Shibata H. Nakano Y. Kitaura Y. Maki M. Biochem. Biophys. Res. Commun. 2002; 291: 1166-1172Crossref PubMed Scopus (58) Google Scholar). Positive clones were sequenced using an automated fluorescent sequencer, ABI PRISM 310 (PE Applied Biosystems). A computer homology search using the advanced BLAST program and coiled-coil region prediction were performed on the World Wide Web. Phylogenetic analysis using Clustal X was performed as described previously (32Ohkouchi S. Nishio K. Maeda M. Hitomi K. Adachi H. Maki M. J. Biochem. (Tokyo). 2001; 130: 207-215Crossref PubMed Scopus (25) Google Scholar). Cell Culture and Transfection—HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C under humidified air containing 5% CO2. One day after HEK293 cells or HeLa cells had been seeded, the cells were transfected with the expression plasmid DNAs by the conventional calcium phosphate precipitation method or by using Fu-GENE6 (Roche Applied Science). After 24 h, cells were harvested and analyzed. To generate HEK293 stable transfectants that constitutively express 3×FLAG-tagged CHMP4b (FLAG-CHMP4b/HEK293), HEK293 cells transfected with the expression vector (pFLAG-CHMP4b) were selected by culturing in the presence of 0.5 mg/ml of G418 for 2 weeks. Expression and Purification of GST-AlixΔC—Escherichia coli BL21 cells were transformed with the plasmid pGST-AlixΔC. The GST fusion protein was purified by binding to glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. GSTAlixΔC was eluted from Sepharose beads with 10 mm reduced glutathione in 50 mm Tris-HCl, pH 8.5, dialyzed against 50 mm Tris-HCl, pH 7.5, and then stored at 4 °C until use. Pull-down Assay—One day after FLAG-CHMP4b/HEK293 (6 × 106) cells had been seeded, the cells were harvested and washed with PBS (137 mm NaCl, 2.7 mm KCl, 8 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.3) and then lysed in 450 μl of lysis buffer (10 mm HEPES-NaOH, pH 7.4, 142.5 mm KCl, 0.2% Nonidet P-40, 0.1 mm pefabloc, 25 μg/ml leupeptin, 1 μm E-64, and 1 μm pepstatin). Supernatants (each 200 μl, obtained by centrifugation at 10,000 × g) were incubated with 10 μg of GST or GST-AlixΔC protein that had been immobilized on glutathione-Sepharose for 3 h at 4 °C. After complexes had been washed three times with the lysis buffer, they were subjected to Western blotting analysis using anti-FLAG monoclonal antibody (mAb). Signals were detected by either the chemiluminescent method using Super Signal West Pico Chemiluminescent Substrate (Pierce) or diaminobenzidine staining. Immunoprecipitation—FLAG-CHMP4b/HEK293 cells transfected with the expression vector of either AlixΔC-V5, GFP-Alix mutants, or Myc-SKD1 were lysed in the lysis buffer and then subjected to centrifugation at 10,000 × g. The supernatants were incubated with anti-V5 mAb, anti-GFP pAb, or anti-FLAG mAb for 1 h and then incubated for 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 either anti-V5 mAb, anti-FLAG mAb, anti-GFP mAb, or anti-Myc mAb. Immunofluorescent Staining—HeLa or HEK293 cells grown on coverslips were fixed in 4% paraformaldehyde/PBS and permeabilized in 0.1% Triton X-100/PBS. After blocking with 0.1% gelatin/PBS, the cells were incubated with primary antibodies either at 4 °C overnight or at room temperature for 1 h and then with secondary antibodies at room temperature for 1 h. Finally, they were mounted with antifading solution (25 mm Tris-HCl (pH 8.7), 10% polyvinyl alcohol, 5% glycerol, 2.5% 1,4-diazobicyclo-(2,2,2)-octane). Immunofluorescences were analyzed under a confocal laser-scanning microscope, LSM5 PASCAL (Zeiss). Epidermal Growth Factor (EGF) Uptake Assay—The transfected HeLa cells were incubated with Dulbecco's modified Eagle's medium for 1 h at 37 °C and then with 0.5 μg/ml tetramethylrhodamine-EGF (Molecular Probes, Inc., Eugene, OR) in 0.5 mg/ml bovine serum albumin/Dulbecco's modified Eagle's medium for 1 h at 4 °C. The cells were washed and incubated in 10% fetal bovine serum/Dulbecco's modified Eagle's medium for 30 min or 6 h at 37 °C. Yeast Two-hybrid Screening—To search for Alix-interacting proteins, the N-terminal region (amino acids 1-660) of human Alix (AlixΔC) that was fused to the Gal4 DNA binding domain was used as bait, and a HeLa cell cDNA library was used as prey in a yeast two-hybrid screen system. Sixty-two positive clones were isolated from 6 × 105 transformants. A search of DNA data bases revealed that the positive clones corresponded to 12 different proteins. Two of them were presumed to be human homologues of yeast Snf7/Vps32 (26Tu J. Vallier L.G. Carlson M. Genetics. 1993; 135: 17-23Crossref PubMed Google Scholar, 27Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (625) Google Scholar) (Fig. 1), and they were also similar to each other (60.7% identity; 136 of 224 amino acid residues). One was identical to HSPC134 (24Zhang Q.-H. Ye M. Wu X.-Y. Ren S.-X. Zhao M. Zhao C.-J. Fu G. Shen Y. Fan H.-Y. Lu G. Zhong M. Xu X.-R. Han Z.-G. Zhang J.-W. Tao J. Huang Q.-H. Zhou J. Hu G.-X. Gu J. Chen S.-J. Chen Z. Genome Res. 2000; 10: 1546-1560Crossref PubMed Scopus (160) Google Scholar), which was referred to as CHMP4 by Howard et al. (25Howard T.L. Stauffer D.R. Degnin C.R. Hollenberg S.M. J. Cell Sci. 2001; 114: 2395-2404Crossref PubMed Google Scholar), and the other was very similar to a hypothetical protein registered in the sequence data bases (DDBJ/GenBank™/EMBL accession number AL050349). In this paper, we designated HSPC134/CHMP4 as CHMP4a and the other as CHMP4b, respectively. Both CHMP4a and CHMP4b were predicted to contain coiled-coil regions (Fig. 1A). Their N-terminal regions are rich in basic residues, whereas their C-terminal regions are acidic. The CHMP4 proteins are part of a large CHMP family (25Howard T.L. Stauffer D.R. Degnin C.R. Hollenberg S.M. J. Cell Sci. 2001; 114: 2395-2404Crossref PubMed Google Scholar). The data base search revealed an additional CHMP4 homologue in this subfamily in the human genome (Fig. 1B). GST Pull-down Assays of AlixΔC and CHMP4b—We performed a GST pull-down assay to determine whether the CHMP4b protein from mammalian cells has the capacity to bind to Alix. First, we constructed several plasmids for expressing various fusion proteins and generated HEK293 cell lines constitutively expressing FLAG-CHMP4b (FLAG-CHMP4b/HEK293). After incubation of the lysate of FLAG-CHMP4b/HEK293 with the GST fusion protein of AlixΔC (GST-AlixΔC) that had been immobilized on glutathione-Sepharose beads, the proteins bound to the beads were subjected to Western blotting using an anti-FLAG mAb as a probe. Whereas CHMP4b did not bind to the negative control GST beads, it bound to the GST-AlixΔC beads (Fig. 2A). Co-immunoprecipitation of CHMP4b and AlixΔC—When the lysate of FLAG-CHMP4b/HEK293 cells transfected with the expression vector of V5-tagged AlixΔC (pAlixΔC-V5) was immunoprecipitated with anti-FLAG mAb, the precipitates contained AlixΔC-V5 as revealed by Western blotting using anti-V5 mAb (Fig. 2B). Moreover, in a reciprocal experiment, FLAG-CHMP4b was also co-precipitated with AlixΔC-V5 using anti-V5 mAb for immunoprecipitation (data not shown). Co-immunoprecipitation of CHMP4b with Alix Segments—To narrow the CHMP4b binding region in AlixΔC, we performed co-immunoprecipitation analysis of FLAG-CHMP4b and GFP fusions of full-length or various truncated forms of Alix. The lysates of FLAG-CHMP4b/HEK293 cells transfected with the expression vectors of GFP-Alix mutants were immunoprecipitated with anti-GFP pAb, and the precipitates were subjected to Western blotting. As shown in Fig. 3, A and B, and summarized in Fig. 3C, we observed interaction of FLAG-CHMP4b with Alix mutants 1-868, 1-660, and 1-423 and weaker interaction with Alix mutant 115-660. No interaction was detected with Alix mutants 1-329, 221-660, and 329-660. Bro1-rhophilin conserved domain located in the N-terminal region of Alix was essential but not sufficient for CHMP4b interaction. Since the Tyr319 of Alix is highly conserved among Alix homologues in various organisms and serves as a potential phosphorylation site for Src-type tyrosine kinases, we replaced Tyr319 of AlixΔC with phenylalanine to examine whether this putative phosphorylation site is needed for the interaction. The interaction between CHMP4b and Y319FΔC was still detectable with a slight decrease in signal. Fluorescence Microscopic Analysis of Overexpressed CHMP4b—We investigated the subcellular localization of CHMP4b using HeLa cells transfected with pFLAG-CHMP4b. Double immunofluorescent staining was performed using an anti-FLAG antibody (Fig. 4, A, E, I, and M) and either an antibody of protein that is a marker of early endosome (EEA1) (Fig. 4B), late endosome and lysosome (Lamp-1) (Fig. 4F), or the trans-Golgi network and late endosome (CI-MPR) (Fig. 4J). We also used GFP-LC3 as an autophagosomal membrane marker instead of staining with an antibody (Fig. 4N). We found that FLAG-CHMP4b was distributed in a punctate manner mainly in the perinuclear area (Fig. 4, A, E, I, and M) and slightly diffused in the cytoplasm. However, only the diffuse pattern was observed in some cells (data not shown). As shown in merged images of the same panels (Fig. 4, C, G, and K) and in images of higher magnification (Fig. 4, D, H, and L), the distribution of FLAG-CHMP4b partly overlapped in the perinuclear region with the distributions of EEA1 and Lamp-1, respectively, in the pFLAG-CHMP4b-transfected cell (Fig. 4, C, left cell, and G, bottom right cell), and the degree of overlapping was greater than that of CI-MPR (Fig. 4K, center cell). On the other hand, GFP-LC3 showed both punctate and diffuse patterns (Fig. 4N, inset), and its fluorescent signals were also detected in the nucleus under the present conditions, in which autophagy was not enhanced by subjecting the cells to nutrient starvation. The distribution of GFP-LC3 was not noticeably changed by overexpression of FLAG-CHMP4b (Fig. 4N) and did not overlap with that of FLAG-CHMP4b in cells expressing both GFP-LC3 and FLAG-CHMP4b (Fig. 4, O and P). To determine whether the distribution of ubiquitinated proteins was affected by CHMP4b overexpression, we examined the localization of ubiquitinated proteins by staining HeLa cells transfected with pCHMP4b-GFP using anti-ubiquitin mAb (FK2) that recognizes mono- and polyubiquitinated proteins but not free monoubiquitin (33Usuba T. Ishibashi Y. Okawa Y. Hirakawa T. Takada K. Ohkawa K. Int. J. Cancer. 2001; 94: 662-668Crossref PubMed Scopus (31) Google Scholar, 34Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (661) Google Scholar). In most of the transfected cells, the distribution of CHMP4b-GFP was essentially similar to that of FLAG-CHMP4b (Figs. 4 and 5A). As shown in Fig. 5B, fluorescent signals of ubiquitinated proteins became stronger in cells expressing CHMP4b-GFP, particularly in the perinuclear area, and showed punctate patterns. Furthermore, ubiquitinated proteins in this area were partially co-localized with CHMP4b-GFP (Fig. 5, C and D). When exogenously expressed FLAG-tagged ubiquitin was monitored, accumulation and partial co-localization of FLAG-tagged ubiquitin with GFP-tagged CHMP4b were similarly observed (data not shown). Accumulation of ubiquitinated proteins in the cells overexpressing CHMP4b suggests that overexpression of CHMP4b may disturb the endosomal sorting pathway. To provide functional data supporting this speculation, we monitored the fate of endocytosed EGF. HeLa cells transfected with pCHMP4b-GFP were incubated with Tetramethylrhodamine-EGF at 4 °C for 1 h, washed, and then allowed to uptake EGF at 37 °C for 30 min or 6 h. Internalized EGF was observed at 30 min, and noticeable change was not detected between the CHMP4b-GFP expressing cells and the untransfected cells (Fig. 5, E-G). At 6 h, fluorescent signals of tetramethylrhodamine-EGF were undetectable in most of the untransfected cells, whereas EGF remained in the perinuclear region of the cells expressing CHMP4b-GFP (Fig. 5, H-J). However, there was a variation in the amount of remaining EGF among
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