Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor

Abstract

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.

Fig. 1.

Induction of hGBP-1 by IFN-γ. (A) hGBP-1 is rapidly induced in the hours after IFN-γ pulse, and the expression level continues to rise through 24 h of IFN-γ induction. Expression shown by immunoprecipitation from HFFs. Note that IFN-γ pulse is followed by 4-h radiolabel. hGBP-1 relocalizes to the Golgi apparatus after AlF treatment in both (B) HeLa M cells and (C) HFFs.

Fig. 2.

Golgi association correlates with membrane localization by cell fractionation. HFF cells were pretreated with IFN-γ for 20 h, then treated for 15 min with AlF at 37°C where indicated. Cells were lysed with a ball-bearing homogenizer and the postnuclear supernatant centrifuged on a sucrose gradient to float the membranes. Twelve fractions were collected and analyzed by SDS/PAGE and Western blotting. Earliest fractions (1-3) are the least dense and contain the membranes.

Fig. 3.

hGBP-1 requires farnesylation for Golgi relocalization. Farnesylation requirement for Golgi relocalization (A). AlF-stimulated relocalization of hGBP-1 to the Golgi does not occur when hGBP-1 is induced in the presence of a farnesyl transferase inhibitor (FTI). Exogenously produced hGBP-1 cannot relocalize without treatment with IFN-γ before AlF exposure (B). Note that in B, Texas red fluorescence corresponds to anti-myc tag staining for exogenous hGBP-1.

Fig. 4.

Characterization of hGBP-1 mutants. (A) GTP-agarose binding for recombinant wild-type hGBP-1 and its mutants. (B) GTP hydrolysis activity of proteins examined in A.(C) Location of hGBP-1 mutants shown on nucleotide-bound crystal structure. R48P in red, T75A in blue, and E99A in green. C589S is not shown on the structure, because the structure is truncated at the C terminus. Its approximate location on the protein is indicated by ^*. Analysis was done on rasmol 2.6 software.

Fig. 5.

Molecular requirements for hGBP-1 recruitment to the Golgi. Neither the farnesyl mutant (A) nor the R48P GTP nonbinding mutant (B) of hGBP-1 can relocalize to the Golgi upon AlF treatment. Both mutants appear to act in a dominant-negative fashion, suppressing the ability of endogenous IFN-induced hGBP-1 to relocalize to the Golgi; fluorescein staining is with rabbit-anti-hGBP1 and reflects both exogenous and endogenous hGBP-1, whereas Texas red staining is for myc-tagged (exogenous) hGBP-1. In C, a mutant that can bind but not hydrolyze GTP (T75A) cannot relocalize to the Golgi without AlF treatment, even with IFN-γ pretreatment. It does relocalize to the Golgi efficiently in the presence of AlF and IFN-γ.