Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.
Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.
Modiano
Nir
N
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8011, USA.
Lu
Yanping E
YE
Cresswell
Peter
P
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D017607:Aluminum Compounds; D015153:Blotting, Western; D002458:Cell Fractionation; D003001:Cloning, Molecular; D051231:Farnesyltranstransferase; D005455:Fluorescent Antibody Technique; D005459:Fluorides; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism* / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000378:metabolism*; D008715:Methionine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D017368:Protein Prenylation / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects; D012190:Retroviridae; D014161:Transduction, Genetic
D017607:Aluminum Compounds; C490287:GBP1 protein, human; C096898:L 744832; D007371:Interferon-gamma; D006160:Guanosine Triphosphate; D008715:Methionine; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride