Intracellular Trafficking of Guanylate-Binding Proteins Is Regulated by Heterodimerization in a Hierarchical Manner

Abstract

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins.

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

“…These speckles form independently of an infection and can be biochemically isolated (9), thus arguing that p62-GBP1/2 complexes are assembled before their translocation to PVs. Additional types of GBPs can be recruited through heterotypic protein-protein interactions between distinct members of the GBP protein family, as such interactions have been described previously (16,18). Moreover, additional ubiquitin-binding proteins like NDP52 may aid in the delivery of GBPs to PVs.…”

“…Binding of GTP results in dimer formation; subsequent GTP hydrolysis prompts conformational changes that enable GBPs to assemble as tetramers (14,15). Mutations in the G domain that reduce nucleotide binding affinities and hydrolytic activity block GBP oligomerization, constrain the localization of GBPs to the cytoplasm, and prevent GBPs from binding to PV membranes (9,(15)(16)(17)(18). These observations support a model in which GBP monomers are diffusely distributed in the cytoplasm and GBP oligomers associate with membranes.…”

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

“…In previous studies IFNγ-induced endogenous hGBP1 has been found in a punctate or vesicular cytoplasmic distribution throughout the whole cell (27). However, the exact assignment of the punctate structures to an intracellular membranous compartment has not been made.…”

“…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).…”

“…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). In the case of murine GBPs, localization to the parasitophorous vacuole of Toxoplasma gondii depends on isoprenylation and a functional LG domain (29).…”