The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1.
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-beta (TbetaRII) receptors (1,2 ). 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 (Leu(489)-Leu(490)) 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.
Lalioti
Vassiliki S
VS
Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Madrid 28049, Spain.
Vergarajauregui
Silvia
S
Pulido
Diego
D
Sandoval
Ignacio V
IV
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D056970:Co-Repressor Proteins; D003902:Detergents / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051275:Glucose Transporter Type 4; D047908:Intracellular Signaling Peptides and Proteins; D007930:Leucine / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D017830:Octoxynol; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D002352:Carrier Proteins; D056970:Co-Repressor Proteins; C491473:Daxx protein, mouse; D003902:Detergents; D051275:Glucose Transporter Type 4; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D025842:SUMO-1 Protein; C495693:Slc2a4 protein, mouse; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D007930:Leucine