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First published online 16 March 2004
doi: 10.1242/jcs.01039

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IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3

Department of Immunology, Max-Planck-Institute for Infection Biology, Schumannstrasse 21-22, 10117 Berlin, Germany

View larger version (20K): [in a new window]   Fig. 1. Yeast two-hybrid interaction between IIGP and mHk3. A, The interaction between IIGP, the GTPase negative mutant IIGP_S83N, controls (lamin, Drosophila Bicoid) or the other members of the 47 kDa GTPase protein family and full-length mHk3 were analyzed by growth on selective medium lacking leucine or for lacZ reporter activity. A Leu+ß-gal+ (Leu+lacZ+) phenotype is indicated by +, while – indicates no detectable reporter activity. (B) Schematic depiction of the domains in mHk3. The N-terminal region (hatched) harbors a microtubule binding domain. The four putative central coiled-coil regions are indicated (dark gray) and the C terminus (dotted) depicts the region involved in Golgi membrane association (Walenta et al., 2001). Truncated mutants of mHk3 were generated and analyzed for interaction with IIGP. ß-Galactosidase activities, reflecting strength of the interactions, were determined by a liquid culture assay and values are given in Miller Units. (C) Schematic depiction of IIGP with the localization of the guanylate-binding domain (black) indicated. Deletion variants of IIGP were tested for interaction with full-length mHk3 by growth on selective medium lacking leucine or for lacZ reporter activity. All analytical experiments were repeated twice with almost identical results.

View larger version (40K): [in a new window]   Fig. 2. Physical interaction of endogenous IIGP and mHk3. Immunoprecipitation of endogenous IIGP or mHk3 from lysates of IFN-stimulated or non-stimulated BMM. (A) The lysates were pre-cleared by immunoprecipitation with isotype matched control mAbs. Subsequently, IIGP was immunoprecipitated with the anti-IIGP mAb 5D9 either prior or after incubation for 30 min at 32°C with addition of GTPS or GDPßS. Purified proteins were subjected to SDS-PAGE and western blotting. Co-immunoprecipitated mHk3 was revealed with an affinity-purified rabbit anti-hook3 serum. (B) The lysates were pre-cleared by use of a non-immune rabbit serum and mHk3 was subsequently purified from lysates of BMM stimulated with IFN or not. IIGP was revealed on western blots using the anti-IIGP mAb 5D9. To determine the total amount of IIGP (B) or mHk3 (A) recovered by specific immunoprecipitation, 1/5 of the samples that were probed for co-purification were analyzed in parallel by western blotting with the respective antibodies.

View larger version (77K): [in a new window]   Fig. 3. Subcellular localization of IIGP and mHk3 in IFN-stimulated BMM. The cells were stained with the anti-IIGP mAb 5D9 revealing the typical distribution of IIGP, which associates with the ER and the Golgi (A,D). The localization of mHk3 was revealed by employing an affinity-purified rabbit anti-hook3 serum (B,E). Cy2- (mHk3) and Cy3- (IIGP) labeled secondary reagents were used. In murine BMM mHk3 strongly accumulated in a juxtanuclear region reminiscent of the position of the MTOC and Golgi. In addition, a reticulate staining for mHk3 was observed in the perinuclear region. Close spatial proximity of IIGP and mHk3, which partially colocalized, was evident in the merged images (C,F).

View larger version (87K): [in a new window]   Fig. 4. Microtubule-dependent localization of IIGP to the Golgi. (A) subfraction of IIGP (A,C) localizes to the Golgi in IFN-stimulated murine BMM revealed by co-localization with the Golgi-resident protein -mannosidase (B,C). Imaging specifically the juxtanuclear accumulation of mHk3 (E,F) reveals co-localization with the cis-Golgi matrix protein GM130 (D,F) as well as nearby accumulation which is reminiscent to the position of the MTOC (data not shown). The IFN-stimulated BMM were treated for 1 hour with nocodazole to disrupt microtubules (G-L). The fine reticulate localization of IIGP in untreated cells (A and Fig. 3), appears contracted upon nocodazole treatment, most probably reflecting the loss of the microtubule-dependent extension of the ER into the cellular periphery (G,I,J,L) (Terasaki et al., 1986). Dispersion of the -mannosidase staining reflects the formation of Golgi-like structures at transitional ER exit sites (H,I) and colocalization with IIGP is not evident (G,I). Likewise, nocodazole treatment induces a scattered distribution of mHk3 staining (K,L).

View larger version (71K): [in a new window]   Fig. 5. Subcellular fractionation of IFN-stimulated BMM. Membrane enriched fractions from post-nuclear supernatants of IFN-stimulated BMM were prepared by separation on a sucrose-step gradient. Fraction I is the 8.5% and fraction II the 20% sucrose layer. The 20-30% and the 30-38% sucrose layer interphases constitute fraction III and IV, respectively. The pellet was designated as fraction V. Golgi and ER membrane-enriched fractions were identified upon SDS-PAGE and western blotting with mAbs against Golgi-associated FTCD and the ER resident protein calnexin. The differential distributions of IIGP, IGTP, mHk3, and -tubulin were analyzed with specific antibodies as described in Materials and Methods.

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