Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells.
Small GTP binding proteins of the rab/YPT family are essential regulators of vectorial transport in the eukaryotic cell. Members of the rab/YPT1 family are found on the cytoplasmic surface of distinct intracellular membrane compartments. Membrane attachment is facilitated by a C-terminal geranylgeranyl moiety. In this report we investigated posttranslational modification and membrane binding of the rab6 protein, a member of the rab/YPT family located on the Golgi apparatus. A set of point mutations, which simulate the GDP or GTP bound conformation, was introduced into the rab6 cDNA. The mutated cDNAs were expressed in insect cells and the ability of the protein products to undergo geranylgeranyl modification and membrane association was assessed by Triton X-114 partition and cell fractionation. We report here that the modification of rab6 in insect cells depends on protein conformation. Only the GDP bound form, but not the GTP bound form is isoprenylated and subsequently membrane bound.
Department for Experimental Tumorbiology, Muenster University, Germany.
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Schiedel
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Barnekow
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Mayer
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009711:Nucleotides / Q000494:pharmacology*; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017368:Protein Prenylation / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism*; D020691:rab GTP-Binding Proteins; D018631:ras Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D009711:Nucleotides; C094135:Rab6 protein; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020691:rab GTP-Binding Proteins; D018631:ras Proteins