The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx
2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês
10.1074/jbc.m110294200
ISSN1083-351X
AutoresVasiliki Lalioti, Silvia Vergarajauregui, Diego Pulido, Ignacio V. Sandoval,
Tópico(s)Fungal and yeast genetics research
ResumoIn this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464–509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-β (TβRII) receptors (1Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 2Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell. Biol. 2001; 3: 708-714Crossref PubMed Scopus (307) Google Scholar). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu489-Leu490) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4. In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464–509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-β (TβRII) receptors (1Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 2Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell. Biol. 2001; 3: 708-714Crossref PubMed Scopus (307) Google Scholar). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu489-Leu490) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4. The cytoplasmic domain of membrane proteins plays important roles in their transport, signal transduction, organization of protein scaffolds, and regulation of their turnover. Trafficking of GLUT4 in adipose and skeletal muscle cells is regulated by insulin and muscle contraction and is critical for the control of glucose levels in blood. Upon increase in insulin levels and muscle contraction the GLUT4 retained in intracellular stores is translocated to the plasma membrane, where it facilitates glucose transport (3James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (517) Google Scholar). Trafficking of GLUT4 is mediated by motifs localized to the amino and carboxyl-cytoplasmic domains of the protein, though their characterization and the identification of the factors involved in their reading is incomplete. SUMO (also called sentrin, PIC1, and GMP1), a 101-amino acid ubiquitin-like modifier protein that is highly conserved from yeast to human, appears to control protein turnover and compartmentalization (4Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (661) Google Scholar). Three members of the SUMO family have been described in vertebrates. They show major structural differences in the sequences of their N-extensions, which are absent in ubiquitin. It has been shown recently that Ubc9, the only E2-type SUMO1-conjugating enzyme described in vertebrates, interacts with the carboxyl-cytoplasmic domain of GLUT4 as part of a mechanism that slows its turnover (5Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (145) Google Scholar). Overexpression of Ubc9 increases GLUT4 abundance 8-fold, probably as result of the conjugation of SUMO1 to GLUT4 and the resistance of the conjugate to degradation (5Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (145) Google Scholar). Interestingly, overexpression of Ubc9 decreases the levels of GLUT1, the ubiquitous glucose transporter homologous to GLUT4, by 2-fold. Ubc9 binds to a highly conserved sequence of 11 amino acids contained in the C-cytoplasmic domains of GLUT4 (RVPETRGRTFD) and GLUT1 (KVPETKGRTFD) (5Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (145) Google Scholar). Here we report that Daxx, 1The abbreviations used are: Daxxdeath-associated proteinPMLpromyelocytic leukemiaJNKc-Jun NH2-terminal kinaseX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideccarboxyl domainLDMlow density membraneHDMhigh density membraneHAhemagglutininPMplasma membraneTβRIItype II TGF-β receptorGLUTglucose transporter an adaptor protein associated with the Fas receptor and TβRII that mediates activation of JNK and cell apoptosis (1Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 2Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell. Biol. 2001; 3: 708-714Crossref PubMed Scopus (307) Google Scholar, 6Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (536) Google Scholar) and is distributed between the PD10 nuclear bodies enriched in SUMOlated proteins (7Pluta A.F. Earnshaw W.C. Goldberg I.G. J. Cell Sci. 1998; 111: 2029-2041Crossref PubMed Google Scholar, 8Everett R.D. Earnshaw W.C. Pluta A.F. Sternsdorf T. Ainsztein A.M. Carmena M. Ruchaud S. Hsu W.L. Orr A. J. Cell Sci. 1999; 112: 3443-3454Crossref PubMed Google Scholar, 9Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T. Strauss 3rd, J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-234Crossref PubMed Scopus (694) Google Scholar, 10Torii S. Egan D.A. Evans R.A. Reed J.C. EMBO J. 1999; 18: 6037-6049Crossref PubMed Scopus (236) Google Scholar, 11Bell P. Brazas R. Ganem D. Maul G.G. J. Virol. 2000; 74: 5329-5336Crossref PubMed Scopus (38) Google Scholar, 12Li H. Leo C. Zhu J., Wu, X. O'Neil J. Park E.J. Chen J.D. Mol. Cell. Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (309) Google Scholar, 13Maul G.G. Negorev D. Bell P. Ishov A.M. J. Struct. Biol. 2000; 129: 278-287Crossref PubMed Scopus (236) Google Scholar) and the cytoplasm (2Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell. Biol. 2001; 3: 708-714Crossref PubMed Scopus (307) Google Scholar, 14Zhong S. Muller S. Ronchetti S. Freemont P.S. Dejean A. Pandolfi P.P. Blood. 2000; 95: 2748-2752Crossref PubMed Google Scholar, 15Zhong S. Salomoni P. Ronchetti S. Guo A. Ruggero D. Pandolfi P.P. J. Exp. Med. 2000; 191: 631-640Crossref PubMed Scopus (195) Google Scholar, 16Ko Y.G. Kang Y.S. Park H. Seol W. Kim J. Kim T. Park H.S. Choi J. Kim S. J. Biol. Chem. 2001; 276: 39103-39106Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), interacts physically with C-GLUT4 but not with C-GLUT1. As GLUT4, a small population of Daxx is conjugated to SUMO1. Microscopy studies localize Daxx to the nucleus and to punctate cytoplasmic structures identified as low density microsomes by cell fractionation studies. The binding of Daxx to GLUT4 is discussed in the framework of their demonstrated SUMOlation. death-associated protein promyelocytic leukemia c-Jun NH2-terminal kinase 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside carboxyl domain low density membrane high density membrane hemagglutinin plasma membrane type II TGF-β receptor glucose transporter EcoRI and BamHI restriction sites were introduced by PCR at positions 1495 and 1748 into the cDNA of GLUT4. The same technique was used to introduce EcoRI and XhoI sites at positions 1552 and 1736 into the cDNA of GLUT1. The cDNA encoding C-GLUT4 (residues 464–509) and C-GLUT1 (residues 443–491) were cloned into the EcoRI/BamHI and EcoRI/XhoI sites, respectively, of the pLexA plasmid and used to study their two-hybrid interactions with Daxx. Subcloning of C-GLUT4 into the HindIII and XhoI sites of the pcDNA3 vector was also performed by PCR. The open reading frame of full-length human Daxx, the kind gift of Dr. A.F. Pluta (University of Maryland), was cloned into the two-hybrid pB42AD plasmid and into the pcDNA3.1H6C vector as described (7Pluta A.F. Earnshaw W.C. Goldberg I.G. J. Cell Sci. 1998; 111: 2029-2041Crossref PubMed Google Scholar). The cDNA encoding N-Daxx (residues 1–572) was cloned into the BamHI and HindIII sites of the pRSETA vector and subcloned into the EcoRI and XhoI sites of the pB42AD plasmid. All the DNA subcloned or amplified by PCR were sequenced before use. The two-hybrid screen of a human heart MATCHMAKER LexA library was performed according to the indications of the manufacturer (CLONTECH, Palo Alto, CA) with minor modifications. Approximately 0.5 mg of the library cDNA was transformed into the yeast strain EGY48 carrying the pLexA:C-GLUT4 plasmid. The first 5 × 106co-transformants were spread on SD/−His−Leu−Trp−Ura plates, and then the Leu+ yeast colonies were spread on SD/Gal/Raf/X-gal/−His−Leu−Trp−Ura plates. Grown blue colonies were isolated for the identification of the library plasmids and further study. Positive library plasmids were transformed for second round into EGY48 to asses the two-hybrid interactions with the pLexA:C-GLUT4 plasmid. The relative strength of the two-hybrid interactions of Daxx with wild-type C-GLUT4 and with the C-GLUT4 mutants was quantified using the liquid β-galactosidase assay as described (17Rose M. Botstein D. Methods Enzymol. 1983; 101: 167-180Crossref PubMed Scopus (282) Google Scholar). The C-GLUT4 mutants developed included C-GLUT4(Arg483-Ala484), C-GLUT4(Ala489-Ser490), C-GLUT4(Ala502) and C-GLUT4Δ5. They were developed by substituting Arg-Ala for the Phe483-Arg484pair, by replacing the pair Ala-Ser for the Leu489-Leu490 pair, by substituting Ala for Tyr502, and by removing the last five C-residues of GLUT4, respectively. EcoRI and BamHI sites were introduced by PCR in all the mutants to clone them into the corresponding sites of the pLexA vector. Full-length DaxxH6C, C-DaxxH6C (residues 661–740), N-DaxxH6C (residues 1–572), and C-GLUT4 were in vitro synthesized and 35S-labeled by the transcription/translation of the pcDNA3.1H6C:Daxx, pcDNA3.1H6C:C-Daxx, and pcDNA3:C-GLUT4 in the TNT rabbit reticulocyte lysate system (Promega). The 35S-labeled Daxx proteins were incubated with 10 μl of the Co2+-based Talon affinity resin (CLONTECH) and then for 60 min at 4 °C in 50 mm Tris, pH 8, 10% glycerol, 250 mm NaCl with or without 35S-labeled C-GLUT4. After washing the resin with 15 mm imidazole, the retained proteins were released by incubation with 200 mm EDTA and then resolved by SDS-PAGE and analyzed by autoradiography. Cells were grown on plastic dishes or glass coverslips. 3T3-L1 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4 mm glutamine, 50 mg/liter streptomycin, 100 IU/liter penicillin and nonessential amino acids (complete medium) at 37 °C in a humidified CO2 incubator. The development of clonal 3T3-L1 stably transfected with wild-type GLUT4, GLUT4(Ala489-Ser490) or GLUT4(Arg483-Arg484) has been described (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar). The clones were cultured in complete medium containing 7.5 μg/ml puromycin. 3T3-L1 adipocytes were differentiated in vitro and cultured in complete medium with or without insulin as required. The fractionation of 3T3-L1 fibroblasts and 3T3-L1 adipocytes into nuclei, low density microsomes (LDM), high density microsomes (HDM), cytosol, and plasma membrane was performed as described (19Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim Biophys Acta. 1983; 763: 393-407Crossref PubMed Scopus (348) Google Scholar). To study the association of Daxx with LDM, the microsomes were resuspended to 1 mg of protein/ml in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% Triton X-114, and 1 mm phenylmethylsulfonylfluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1.5 μm pepstatin A and, after their incubation for 3 min at 30 °C, overlaid onto a 6% sucrose cushion and centrifuged 3 min at 300 × g to separate the detergent and the aqueous phases (20Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). The two phases as well as the sucrose interphase were analyzed for their content in Daxx by Western analysis using the rabbit polyclonal anti-Daxx M-112 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The rabbit polyclonal (M-112; lot J629) and monoclonal (H-7) antibodies raised against C-Daxx (residues 627–739) were purchased from Santa Cruz Biotechnology, Inc. The mouse monoclonal anti-HA 16B12 antibody and the anti-SUMO1 monoclonal antibodies were purchased from BabCo (Berkeley, CA) and Zymed Laboratories Inc. (San Francisco, CA), respectively. The monoclonal anti-GLUT4 1F8 antibody was from Biogenesis (Poole, UK). The rabbit polyclonal antibodies against C-GLUT4 and the endoplasmic reticulum marker, protein disulfide isomerase, were the kind gift of Dr. G. Holman (Bath University, Bath, UK) and Dr. J. Gonzalez Castaño (Universidad Autónoma de Madrid, Madrid, Spain), respectively. 3T3-L1 fibroblasts and 3T3-L1 adipocytes were lysed for 1 h at 4 °C in 20 mm Hepes, pH 7.5, containing 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 10% glycerol, 20 mm β−glycerophosphate, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 1.5 μm pepstatin A (buffer A). Immunoprecipitations were performed by incubating for 4 h at 4 °C the postnuclear supernatants, developed by a 5-min centrifugation at 600 × g, or the HDM fraction with the corresponding antibodies. The protein-antibody complexes were collected on protein G-Sepharose and washed four times with buffer A containing 0.5% Nonidet P-40 and once with 0.1% SDS in buffer A. The cleaned immunoprecipitates were resolved by SDS-PAGE, using small-size gels (8.5 × 6.5 cm, 0.5 mm thick) or large-size gels (16 × 17 cm, 1.5 mm thick) and then blotted onto nitrocellulose, and the proteins were visualized with specific antibodies using the enhanced chemiluminescence technique. Cells grown for more than 60 h on coverglasses in complete medium were either fixed with cold (−20) methanol or sonicated in cold KHMgE buffer (70 mm KCl, 30 mm Hepes, 5 mm MgCl2, 3 mm EGTA, pH 7.5) to yield plasma membrane lawns. Cells and plasma membrane lawns were single- or double- immunostained with the rabbit polyclonal anti-Daxx M-112 antibody and the mouse monoclonal anti-GLUT4 1F8 antibody as described (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar). The study of protein distribution was performed by confocal microscopy using a Bio-Rad Radiance 2000 microscope with the argon (488 nm) and helio/neon (543 nm) lasers set to 3 and 100, respectively, and the iris aperture set to optimum. We used the yeast two-hybrid system to identify proteins that interact physically with C-GLUT4, the carboxyl-cytoplasmic domain (residues 464–509) of the insulin-sensitive glucose transporter, GLUT4. For this purpose we used a LexA:C-GLUT4-based interaction trap assay to screen a MATCHMAKER LexA cDNA library prepared from human heart, one of the three tissues in which GLUT4 is confined in mammalian cells. We identified three identical C-terminal GLUT4-interacting clones that encoded the Daxx domain comprised between residues 661 and 740, henceforth referred as C-Daxx. The interaction between C-Daxx and C-GLUT4 was further assessed by co-introducing the purified pLexA:C-GLUT4 and pB42AD:C-Daxx plasmids into the yeast strain EGY48 and then immunoprecipitating the HA-tagged C-Daxx with an anti-HA antibody (Fig. 1A). The analysis of the immunoprecipitate with an anti-GLUT4 antibody by Western showed that the pull-down of C-Daxx brought down C-GLUT4. A control experiment run with yeast transformed with pLexA:C-GLUT4 showed that C-GLUT4 was not precipitated by the anti-HA antibody (Fig. 1A). Together these results were confirmatory of the two-hybrid interaction between Daxx and C-GLUT4. The interaction between C-Daxx and C-GLUT4 was of interest since it has been shown that both interact with the SUMO-conjugating enzyme Ubc9 (5Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (145) Google Scholar,21Ryu S.W. Chae S.K. Kim E. Biochem. Biophys. Res. Commun. 2000; 279: 6-10Crossref PubMed Scopus (46) Google Scholar). In addition, Daxx interacts with SUMO1 in BOSC23 cells (21Ryu S.W. Chae S.K. Kim E. Biochem. Biophys. Res. Commun. 2000; 279: 6-10Crossref PubMed Scopus (46) Google Scholar), and it has been reported that some GLUT4 could be conjugated to SUMO1 in 3T3-L1 adipocytes (5Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (145) Google Scholar). The ability of Daxx to interact with C-GLUT4 was further assayed through a biochemical assay. C-Daxx, N-Daxx (residues 1–572), and full-length Daxx tagged with His6 were in vitro translated and labeled with [35S]methionine/cysteine. Samples of each Daxx product were first incubated with 10 μl of a Co2+-based Talon resin and then with 35S-labeled C-GLUT4 to pull down the His-tagged protein complexes. The products retained by the resin were eluted and studied by autoradiography. The results showed that full-length Daxx and C-Daxx (Fig. 1B) but not N-Daxx (data not shown) interacted with C-GLUT4. Furthermore, the coincidence between the sizes of full-length Daxx, C-Daxx, and GLUT4 calculated from their electrophoretic mobility and from their amino acid sequences excluded their modification during their translation in vitro and showed that the interaction between Daxx and C-GLUT4 requires no prior modification of the proteins. C-GLUT4 contains a few transport motifs required for the intracellular sorting and endocytosis of GLUT4 (22Czech M.P. Chawla A. Woon C.W. Buxton J. Armoni M. Tang W. Joly M. Corvera S. J. Biol. Chem. 1993; 1: 127-135Google Scholar). Among these motifs are: the dileucine-based motif (23Verhey K.J. Birnbaum M.J. J. Biol. Chem. 1994; 269: 2353-2366Abstract Full Text PDF PubMed Google Scholar), which mediates the targeting of GLUT4 from the trans-Golgi network to the pericentriolar storage compartment (PC-GSC) (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar) as well as its surface internalization by endocytosis (24Corvera S. Chawla A. Chakrabarti R. Joly M. Buxton J. Czech M.P. J. Cell Biol. 1994; 126: 979-989Crossref PubMed Scopus (103) Google Scholar); and clusters of acidic residues (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar, 25Shewan A.M. Marsh B.J. Melvin D.R. Martin S. Gould G.W. James D.E. Biochem. J. 2000; 350: 99-107Crossref PubMed Scopus (85) Google Scholar), including the last five C-residues that together with the adjacent Tyr502 play a role in the retention of GLUT4 in the PC-GSC (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar). To study if any of these motifs were involved in the interaction of C-GLUT4 with C-Daxx, they were separately inactivated (see Fig. 2). The dileucine-based motif,484RR XXXLL490 (26Sandoval I.V. Martinez-Arca S. Valdueza J. Palacios S. Holman G.D. J. Biol. Chem. 2000; 275: 39874-39885Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), was inhibited by introducing an Ala between the two Arg (GLUT4Arg483-Ala-484 mutant), and by replacing the pair Leu489-Leu490 by the pair Ala-Ser (GLUT4Ala489-Ser490 mutant). The motifs involved in the retention of GLUT4 in the PR-GSC were inhibited by replacing an Ala for Tyr502 (GLUT4Ala502mutant) and by deleting the last five PDEND residues (GLUT4Δ5 mutant). The wild-type C-GLUT4 and the C-GLUT4 mutants were then cloned into the pLexA plasmid and introduced into yeast EGY48 cells previously transformed with the Daxx:pB42AD plasmid. The relative strength of two-hybrid interactions was quantified with the liquid β-galactosidase assay (Fig. 2). The interaction of C-Daxx with C-GLUT4 was significantly inhibited, 2-fold, by the substitution of the Leu489-Leu490 pair by Ala-Ser, but none of the other three mutants developed significantly affected the interaction. We also measured the ability of Daxx to interact physically with GLUT4 in clonal 3T3-L1 fibroblasts stably transfected with HA-GLUT4 and expressing levels of the transporter comparable with those measured in 3T3-L1 adipocytes and adipose tissue (Fig. 3) (18Martinez-Arca S. Lalioti V.S. Sandoval I.V. J. Cell Sci. 2000; 113: 1705-1715Crossref PubMed Google Scholar). For this purpose, postnuclear lysates were separately incubated with a monoclonal antibody against the HA tag introduced in GLUT4 and with a polyclonal antibody against Daxx, and the content of Daxx and GLUT4 in the immunoprecipitates was measured by Western using specific antibodies (Fig. 3). We observed that the immunoprecipitation of GLUT4 with the anti-Ha antibody brought down a small amount of Daxx (Fig. 3A). Furthermore, Daxx was not immunoprecipitated when the incubation was repeated using a lysate from 3T3-L1 fibroblasts that were not expressing GLUT4 (Fig. 3A). Moreover, the immunoprecipitation of Daxx with an anti-Daxx antibody also brought down a small amount of GLUT4 (Fig. 3A). Mock immunoprecipitations performed by incubating lysates from fibroblasts transfected with GLUT4 with a monoclonal antibody against the nuclear antigen NA or with rabbit preimmune serum brought down neither GLUT4 nor Daxx (Fig. 3). It was interesting that lysates and immunoprecipitates contained two GLUT4 species of 64 kDa and 61 kDa. The 64-kDa species was dominant and was preferentially immunoprecipitated by the anti-HA antibody (Fig. 3A). When the above experiment was repeated using 3T3-L1 adipocytes and monoclonal antibodies to stain the proteins (Fig. 3B), the results obtained were comparable with those in fibroblasts, and small amounts of GLUT4 and Daxx were reproducibly found in pull downs of Daxx and GLUT4, respectively. Again, a mock immunoprecipitation performed with rabbit preimmune serum brought down neither Daxx nor GLUT4 (Fig. 3B). Stimulation of adipocytes with 100 nminsulin for 20 and 40 min did not change the amount of GLUT4 pull down by the anti-Daxx antibody or the amount of Daxx immunoprecipitated by the anti-GLUT4 antibody (Fig. 3C). The liquid β-galactosidase assay was also used to study the two-hybrid interaction between C-Daxx and C-GLUT1, the ubiquitous glucose transporter homologous to GLUT1. This was of interest since C-GLUT1 as C-GLUT4 interacts with Ubc9 and appears to be SUMOlated. The two-hybrid assay showed that C-GLUT1 did not interact physically with C-Daxx (Fig. 2). Taken together this result and the results of the C-GLUT4 experiments showed that the interaction between C-Daxx and C-GLUT4 was specific. Furthermore, this observation also excludes the fact that the 11-residue-long Ubc9 domain shared by C-GLUT4 and C-GLUT1 is involved in the interaction of Daxx with C-GLUT4. The cellular distribution of Daxx is controversial. Most published studies indicate that Daxx is entirely nuclear (7Pluta A.F. Earnshaw W.C. Goldberg I.G. J. Cell Sci. 1998; 111: 2029-2041Crossref PubMed Google Scholar, 8Everett R.D. Earnshaw W.C. Pluta A.F. Sternsdorf T. Ainsztein A.M. Carmena M. Ruchaud S. Hsu W.L. Orr A. J. Cell Sci. 1999; 112: 3443-3454Crossref PubMed Google Scholar, 9Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T. Strauss 3rd, J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-234Crossref PubMed Scopus (694) Google Scholar, 10Torii S. Egan D.A. Evans R.A. Reed J.C. EMBO J. 1999; 18: 6037-6049Crossref PubMed Scopus (236) Google Scholar, 11Bell P. Brazas R. Ganem D. Maul G.G. J. Virol. 2000; 74: 5329-5336Crossref PubMed Scopus (38) Google Scholar, 12Li H. Leo C. Zhu J., Wu, X. O'Neil J. Park E.J. Chen J.D. Mol. Cell. Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (309) Google Scholar, 13Maul G.G. Negorev D. Bell P. Ishov A.M. J. Struct. Biol. 2000; 129: 278-287Crossref PubMed Scopus (236) Google Scholar). However, recent studies have traced Daxx to the cytoplasm and showed that its distribution between cytoplasm and nucleus can be regulated (2Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell. Biol. 2001; 3: 708-714Crossref PubMed Scopus (307) Google Scholar, 14Zhong S. Muller S. Ronchetti S. Freemont P.S. Dejean A. Pandolfi P.P. Blood. 2000; 95: 2748-2752Crossref PubMed Google Scholar, 15Zhong S. Salomoni P. Ronchetti S. Guo A. Ruggero D. Pandolfi P.P. J. Exp. Med. 2000; 191: 631-640Crossref PubMed Scopus (195) Google Scholar, 16Ko Y.G. Kang Y.S. Park H. Seol W. Kim J. Kim T. Park H.S. Choi J. Kim S. J. Biol. Chem. 2001; 276: 39103-39106Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The interaction of Daxx with C-GLUT4 led us to compare the cellular distributions of Daxx and GLUT4 in cellular fractions (Figs.4 and 5) and by microscopy (see Fig. 7). Fractions of 3T3-L1 fibroblasts and 3T3-L1 adipocytes enriched in nuclei, cytoplasm, HDM, and LDM were scrutinized for Daxx using the anti-Daxx polyclonal antibody. We detected high levels of Daxx in the nucleus and LDM (Fig. 4,A and B). The absence of Daxx and NA-1 (a nuclear protein that, homogeneously distributed through the nucleoplasm, becomes associated with the chromatin upon its condensation) from the cytosol and HDM fractionated from fibroblasts discarded the fact that the Daxx detected in LDM was produced by contamination with the protein released from broken nuclei. It remains unclear if the traces of Daxx in HDM isolated from 3T3-L1 adipocytes were produced by the contamination of HDM with LDM. In contrast with Daxx, GLUT4 was detected in HDM, LDM, and plasma membrane (Fig. 4B).Figure 5Insulin stimulation does not change the confinement of Daxx in LDM distribution of distinct high molecular GLUT4 species among HDM and LDM. HDM, LDM, and PM fractions were prepared from 3T3-L1 adipocytes incubated for 3 h in Dulbecco's modified Eagle's medium (a), before further incubation for 20 min (b), and 40 min (c) with 100 nm insulin. The fractions were manipulated as described in the legend to Fig. 4, and their content in Daxx and GLUT4 was studied by Western using specific polyclonal antibodies. The Westerns of the LDM and HDM fractions stained for GLUT4 were digitally treated to facilitate the study of the changes in the levels of GLUT4 after insulin stimulation. Asterisks mark the position of the 90-kDa GLUT4 species in the Westerns.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Double immunostaining of 3T3-L1 adipocytes with anti-Daxx and anti-GLUT4 antibodies.In vitro differentiated adipocytes cultured in complete medium (A–I, K) and plasma membrane lawns (J) were fixed-permeabilized with cold (−20 °C) methanol. Daxx and GLUT4 were stained with the rabbit polyclonal M-112 antibody (fluorescein channel) and the mouse monoclonal antibody 1F8 (Texas Red channel), respectively. Nuclei from 3T3-L1 adipocytes (K) and 3T3-L1 fibroblasts (L) grown in complete medium were stained for Daxx with antibody M-112 (fluorescein channel). The stained cells were stu
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