Direct interaction of Rab4 with syntaxin 4.
In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.
Li
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Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.
Omata
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W
Kojima
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Shibata
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H
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation; D002478:Cells, Cultured; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D050765:Qa-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050827:Syntaxin 1; D013873:Thionucleotides / Q000494:pharmacology; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D007328:Insulin; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D020695:rab4 GTP-Binding Proteins