Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-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 Ras͞Rab 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-␥-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-␥. 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.antiviral proteins ͉ GTPases ͉ innate immunity

Human Guanylate Binding Protein-1 Is a Secreted GTPase Present in Increased Concentrations in the Cerebrospinal Fluid of Patients with Bacterial Meningitis

“…In this framework a recent report on Golgi localization of hGBP-1 in cells co-stimulated with IFN-␥ and AlF suggested that hGBP-1 may be classically secreted. 13 However, under physiological conditions without AlF, no significant Golgi enrichment of hGBP-1 was detected. This does not exclude that Golgi translocation may be important for certain biological functions of hGBP-1, but clearly indicates that the amount of hGBP-1 in the Golgi may be too small to cause the release of significant amounts of hGBP-1 via the classical route.…”

“…12 Recently, it has been demonstrated in HeLa cells that hGBP-1 can translocate to the Golgi membranes. 13 The translocation process required the GTPase activity of hGBP-1, stable induction of a protein structure resembling the GTP-bound form [induced by aluminum fluoride (AlF)], a functional isoprenylation signal, and IFN-␥ stimulation of the cells. 13 Presence in vesicles and/or Golgi localization are hallmarks of secreted proteins.…”

Nucleotide-dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate-binding protein 1

“…For both functions, the ability to interact with cellular membranes is a prerequisite. In the case of hGBP1 and murine GBP2 (mGBP2), abolishing the C-terminal lipid modification results in biologically inactive proteins (26)(27)(28)33). To understand the function and mechanism of hGBP1 on a molecular level, it is therefore of critical importance to study the protein in the lipid modified state.…”

“…In IFN-induced cells, hGBP1 is localized in cytosolic puncta, unidentified to date (26,27). The addition of AlFx induces redistribution of hGBP1 to the Golgi complex in a farnesylation-dependent manner (26,28). On the other hand, mutation of the contact between the LG domain and the α12/13 domain results in the localization of hGBP1 at the plasma membrane, whereas a protein mutant impaired in binding nucleotides displays a purely cytosolic staining (12,27).…”

Entry of diabetogenic T cells into islets induces changes that lead to amplification of the cellular response

“…The p47 family members are recruited to nascent phagosomes after infection with bacteria and parasites (15). The p65 family member Gbp1 is recruited to the Golgi after chemical activation (20). The potential biological outcome of p47 and p65 activation during microbial infection has been postulated to involve rapid killing/ degradation and trafficking of the pathogen to the antigenprocessing compartments.…”