ARTICLE |
Correspondence to: Erik C. Böttger, Inst. für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 8, D-30625 Hannover, Germany.
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Summary |
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IFP 35 is an interferon (IFN)-regulated leucine zipper protein, expression of which is observed in a variety of cell types including monocytes/macrophages, epithelial cells and fibroblasts. Using immunofluorescence studies, we demonstrate that IFP 35 is found in characteristic punctate cytoplasmic structures after IFN treatment. Co-localization experiments using double immunofluorescence and confocal laser scanning microscopy failed to show association of IFP 35 with known organelles (mitochondria, peroxisomes, endoplasmic reticulum, lysosomes, endosomes, Golgi complex), ribosomes, or actin filaments. Subcellular fractionation to separate membrane-associated from cytoplasmic proteins demonstrated that IFP 35 localizes to the cytoplasm. Separation of postnuclear supernatant from HeLa cells by gel filtration revealed that IFP 35 eluted at a molecular mass of 200440 kD, suggesting that IFP 35 is part of protein complexes. Electron microscopic studies showed cytoplasmic clusters of a few aggregates of IFP 35 in IFN-treated cells which were neither associated with nor surrounded by a membrane. A combination of immunoprecipitation and immunofluorescence studies of cells transfected with a hemagglutinin epitope-tagged IFP 35 expression construct demonstrated complex formation and co-localization of endogenous and transfected IFP 35. Taken together, our studies demonstrate that IFP 35 associates with unique cytoplasmic structures that are distinct from known organelles and resemble large protein aggregates. (J Histochem Cytochem 47:169182, 1999)
Key Words: interferon, IFP 35, leucine zipper, cytoplasmic structure, protein aggregates, organelles
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Introduction |
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Interferons (IFNs) are a class of proteins with diverse biological functions. These pleiotropic effects include the ability to inhibit viral replication, to restrict growth of tumor cells in vitro and to regulate immunologically important functions of multiple cell types (
The leucine zipper motif consists of a periodic repetition of leucines or other hydrophobic residues forming a parallel, two-stranded, -helical coil (
As an approach to understanding the function of IFP 35, we sought to characterize its subcellular localization. In the cytoplasm of eukaryotic cells, different compartments have been defined by morphological criteria. Prominent organelles are mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, endosomes, lysosomes, and various kinds of trafficking vesicles that are involved in endocytic and exocytic pathways. In addition to organelles, eukaryotic cells are characterized by an internal skeleton, the cytoskeleton, that gives the cell its shape, its capacity to move, and the ability to arrange and transport its organelles. Cytoplasmic proteins, which are not compartmentalized by association with vesicles, organelles, or the cytoskeleton, are in general distributed homogeneously within the cytoplasm. Notably, some rare examples of cytoplasmic proteins with a nondiffuse cytoplasmic distribution have been described, i.e., nonnuclear localization of the human p53 tumor suppressor protein appearing as punctate cytoplasmic structures (
This report describes the results of a variety of techniques, including immunofluorescence microscopy, immunochemistry, subcellular fractionation, and electron microscopy, examining the intracellular distribution of IFP 35. Our results demonstrate that IFP 35 distributes in a unique IFN-induced punctate cytoplasmic pattern that is suggestive of large protein structures.
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Materials and Methods |
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Cell Culture and Immunofluorescence Microscopy
HeLa S3 (Deutsche Sammlung von Mikroorganismen und Zellkulturen; Braunschweig, Germany), GM637 (. All culture media contained 862 mg/liter L-alanyl-L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin (Sigma), and 10% fetal calf serum (Life Technologies).
Cycloheximide (CHX, Sigma; 50 mg/ml stock solution in ethanol) was added to a final concentration of 50 µg/ml: this concentration inhibits protein synthesis in HeLa cells by greater than 95% as determined by [35S]-cysteine incorporation. Nocodazole and cytochalasin D [stock solutions of 50 mM and 10 mM, respectively, prepared in dimethylsulfoxide (DMSO)] were diluted to the following final concentrations in culture medium: 50 µM, 25 µM, and 2.5 µM for nocodazole and 10 µM, 5 µM, and 2 µM for cytochalasin D. To control for unspecific effects of DMSO, cultures were treated with DMSO in the appropriate concentrations.
For immunofluorescence studies, cells were suspended at 14 x 104 cells/ml in culture medium and added to clear glass coverslips (12-mm diameter; Assistent, Sondheim/Rhön, Germany) placed in 24-well tissue culture plates (1000 µl/well), THP-1 cells were incubated with 20 nM PMA for 24 hr before IFN induction. The following day, cells were treated with 1000 U/ml interferon- (Bioferon, 2 x 107 U/mg protein; Rentschler, Laupheim, Germany) (kindly provided by P.v. Wussow).
According to the different antibodies used in the double immunofluorescence experiments, cells were fixed and permeabilized at room temperature (RT) as follows. For anti-mitochondrial antigen (BioGenex; San Ramon, CA), anti-KDEL peptide (kindly provided by J. Wehland; Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany), and anti-transferrin receptor antibodies (Oncogene; Uniondale, NY), cells were fixed for 10 min with 3% paraformaldehyde in PBS and permeabilized for 15 min with 0.2% Triton X-100. For the anti-S3a antibody (kindly provided by J. Stahl; Max-Delbrück-Centrum, Berlin, Germany), cells were fixed for 30 min with 3% paraformaldehyde and permeabilized for 15 min with 0.2% Triton X-100. For anti-cathepsin D (kindly provided by K. v. Figura; Universität Göttingen, Göttingen, Germany, and also obtained from Oncogene) and anti-mannose 6-phosphate receptor antibodies (kindly provided by K. v. Figura and by B. Hoflack, European Molecular Biology Laboratory, Heidelberg, Germany), cells were fixed for 45 min with 3% paraformaldehyde. For anti-catalase (Calbiochem; Bad Soden/Taunus, Germany), and anti-ß COP antibodies (Sigma), cells were treated for 10 min with ice-cold methanol and 1 min with ice-cold acetone. For staining of IFP 35, two antibodies were used: a polyclonal rabbit antiserum (generated against an IFP 35 maltose binding fusion protein) (
Fixed and permeabilized cells were rinsed three times with PBS and incubated with blocking buffer (0.1% saponin/10% FCS/PBS) for 1030 min at RT, followed by incubation with the appropriate primary antibodies diluted in blocking buffer for 3060 min at RT. After three washes with PBS and one wash with blocking buffer, cells were incubated with secondary antibodies [goat anti-rabbit, goat anti-mouse, donkey anti-rabbit conjugated to fluorescein isothiocyanate (FITC); goat anti-rabbit, goat anti-mouse, donkey anti-goat conjugated to tetramethylrhodamine isothiocyanate (TRITC); all obtained from Jackson ImmunoResearch, West Grove, PA]. Cells were washed three times with PBS, once with blocking buffer followed by three washes with PBS, and finally embedded using Mowiol (Calbio-chem) in PBS, pH 7.4, containing 50 mg/ml 1,4-diazabicyclo[2.2.2.]octane (DABCO; Sigma) to stabilize fluorescence of FITC (for mannose 6-phospate receptor and cathepsin D, 0.5% saponin/PBS was used instead of 0.1% saponin/10% FCS/PBS throughout the procedure). Actin was visualized by staining with 5 µg/ml FITC-conjugated phalloidin (Sigma) for 60 min. Conventional epifluorescence was examined using a Zeiss Axioskop microscope equipped with a Plan-Neo-fluar x63 objective (Zeiss; Oberkochen, Germany) and appropriate filters. Laser scanning microscopy was performed with a BioRad MRC1024 system (BioRad; München, Germany) using an argon/krypton laser and an E2/UBHS filter set. Optical sections were obtained at 0.25-µm intervalls and the combined Z-series were printed on a Tektronix Phaser 440 (Tektronix; Köln, Germany).
Electron Microscopy
HeLa cells were seeded on 6-cm plastic dishes at a density of 5 x 105 cells/dish and induced the following day with 1000 U/ml IFN- for 24 hr. Cells were then washed three times with PBS. Cells were fixed directly on Petri dishes with 0.2% glutaraldehyde and 0.5% formaldehyde (final concentration) in PBS for 1 hr on ice. After three washing steps using PBS containing 0.01% glycine for quenching of free aldehyde groups, cells were scraped from the Petri dishes and embedded in 1.5% agar in PBS. After solidification, small cubes were cut and samples embedded in LR White resin as follows. Sample cubes were dehydrated with 30, 50, 70, and 90% ethanol for 30 min each step on ice, then infiltrated with a 2:1 mixture of LR White resin [containing 1.5 µl accelerator (0.5% benzoylperoxide)/1 ml resin] and 90% ethanol for 1 hr on ice, followed by pure resin given two changes for 1 hr each. Samples were left in a third change overnight and subsequently placed in gelatin capsules with fresh resin and polymerized at 50C for 24 hr. Alternatively, samples were embedded according to the progressive lowering of the temperature (PLT) method by using Lowicryl K4M resin (
Subcellular Fractionation
Cells (7.5 x 106 in 175-cm2 flasks) were washed with PBS and detached using trypsin/EDTA (Sigma). Cells were sedimented and washed three times with PBS. Subsequent steps were carried out at 4C. The cell pellet was diluted to a density of 1.5 x 107 cells/ml in 0.25 M sucrose supplemented with 0.1 mM PMSF and 10 µg/ml aprotinin and homogenized on ice in a Dounce homogenizer with a tight-fitting pestle. Cell disruption was evaluated by phase-contrast microscopy. Nuclei and unbroken cells were removed by centrifugation at 800 x g for 10 min. Postnuclear supernatant (750 µl) was layered on 10-ml Percoll prepared in 0.25 M sucrose and centrifuged for 1.5 hr at 27,000 x g (JA 20; Beckman Instruments, Palo Alto, CA). Fractions of 1 ml were collected starting at the top. Each fraction was run on SDS-PAGE and analyzed by immunoblotting.
Gel Chromatography
Cells were centrifuged at 100 x g for 10 min at RT and then washed three times with PBS. The following steps were carried out at 4C. Cells were diluted in PBS or intracellular buffer (ICB: 3 mM NaCl, 140 mM KCl, 11 mM EGTA, 10 mM Hepes, pH 7.2, adjusted with KOH) to a density of 1 x 106 cells/ml and homogenized using a Dounce homogenizer. Cell debris and nuclei were removed by centrifugation at 800 x g for 10 min. For ICB, MgCl2 was added to final concentration of 1 mM and the postnuclear supernatant incubated on ice for 10 min. Postnuclear supernatant was filter-sterilized through a 0.22 µM filter and 100 µl of the filtrate was separated on a Superose 6 column (HR 10/30; Pharmacia, Uppsala, Sweden) using fast-performance liquid chromatography (FPLC, Pharmacia). Depending on the experiment, the column was equilibrated and eluted by using PBS or ICB/MgCl2 at a flow rate of 0.25 ml/min. Fractions of 0.5 ml were collected and analyzed. The column was calibrated with molecular mass markers (Sigma) [thyroglobulin (669 kD), apoferritin (443 kD), ß-amylase (200 kD), yeast alcohol dehydrogenase (150 kD), albumin (66 kD), bovine carbonic anhydrase (29 kD)].
Preparation of Crude Nuclear and Cytoplasmic Extracts
Cells (1 x 107 in 175-cm2 flasks) were washed with PBS and detached using trypsin/EDTA (Sigma). Cells were sedimented and washed twice with PBS. Subsequent steps were carried out at 4C. The cell pellet was diluted to a density of 1 x 107 cells/500 µl in NP-40 buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40), incubated for 10 min on ice, and centrifuged for 5 min at 800 x g. The supernatant was recentrifuged for 15 min at 15,000 x g and the resulting supernatant was used as crude cytoplasmic extract. The pellet from the low-speed centrifugation was diluted in Buffer C (20 mM HEPES, pH 7.8, 420 mM NaCl, 3 mM MgCl2, 0.2 mM EDTA, 25% glycerin, 1 mM DTT, 0.1 mM PMSF, 10 µg/ml aprotinin) to a concentration of 1 x 107 cells/75 µl, incubated for 45 min on ice with vortexing every 15 min, and centrifuged for 5 min at 15,000 x g. The resulting supernatant served as source for crude nuclear extract. Equal amounts of each fraction were separated by SDS-PAGE (
Immunoblotting
Samples were diluted with SDS sample buffer, denatured, and separated by SDS-PAGE. After electrophoresis proteins were transferred on nitrocellulose membranes (Schleicher & Schuell; Dassel, Germany) by electroblotting (1 hr at 0.8 mA/cm2). Membranes were blocked with PBS containing 0.1% Tween 20 and 5% nonfat milk and then incubated for 1 hr with primary antibodies in PBS containing 0.1% Tween 20 and 1.5% nonfat milk. Membranes were washed three times with 0.1% Tween 20 in PBS and incubated with peroxidase-conjugated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat antibodies (Jackson ImmunoResearch) for 1 hr, depending on the primary antibody used. Visualization was done by chemiluminescence (ECL; Pharmacia).
Immunoprecipitation
HeLa cells were plated in six-well plates at a density of 2 x 105 cells/well, transfected as described below, and induced for 24 hr with 1000 U/ml IFN-. Cells were then washed with PBS, detached using trypsin/EDTA, sedimented, and washed twice with PBS. Cell lysis was performed using 1% NP-40 in PBS as lysis buffer. The cells were incubated for 45 min on ice at a concentration of 2 x 107 cells/ml in lysis buffer and then centrifuged for 5 min at 15,000 x g at 4C. A total of 100 µl of the resulting supernatant was used for immunoprecipitation: 1 µg of anti-HA antibody clone 12CA5 (Boehringer; Mannheim, Germany) was added and incubated for 1 hr at 4C under mild shaking. Then 100 µl of 20% protein ASepharose (Sigma) in lysis buffer was added and incubated for 1 hr at 4C, followed by centrifugation at 350 x g for 5 min at 4C. The resulting pellet was washed four times with lysis buffer and resuspended in 20 µl of SDS sample buffer; 10 µl was separated by SDS-PAGE as described above.
Transfection of HeLa Cells
Cells were plated on six-well plates for preparing cell extracts or on 24-well plates covered with glass coverslips for immunofluorescence studies (2 x 105 or 5 x 104 cells/well, respectively). Cells were cultured overnight and transfected with SuperFect reagent (Qiagen; Hilden, Germany) according to the manufacturer's instructions. Briefly, plasmid DNA (4 µg) was diluted with Dulbecco's MEM containing 4500 mg/ml glucose to a final volume of 100 µl, and 10 µl SuperFect reagent was added. The sample was vortexed for 10 sec, incubated for 1030 min at RT and 600 µl culture medium was added. Cells of a six-well plate were washed twice with PBS, PBS was aspirated thoroughly, and the transfection reagent was added, followed by incubation for 1018 hr at 37C (for transfection of cells grown in a 24-well plate 1 µg DNA was diluted to a volume of 60 µl with Dulbecco's MEM; 5 µl SuperFect reagent and 350 µl of culture medium were subsequently added). After transfection, cells were washed once with PBS and incubated in culture medium supplemented with 1000 U/ml IFN- for 24 hr. Cells were prepared for cell extract preparation, immunoprecipitation, or immunofluorescence studies as described above.
The following vectors, which have been described previously (
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Results |
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Localization of IFP 35 by Immunofluorescence
HeLa cells were induced with IFN and processed for immunostaining using monoclonal anti-IFP 35 as primary antibody. IFN treatment led to an enlargement in both size and number of cytoplasmic structures stained by anti-IFP 35 antibody. Whereas control cells incubated in medium rarely showed punctate structures, IFN-treated cells showed a speckled staining pattern with 1020 larger, irregularly shaped elements distributed throughout the cell. In addition to these structures, a relatively diffuse cytoplasmic staining was observed (Figure 1A and Figure 1B). Sometimes tinier structures of more uniform size were also observed. Treatment with IFN- resulted in the same speckled pattern (data not shown). A polyclonal anti-IFP 35 antiserum as primary antibody gave identical immunofluorescence results with somewhat higher background staining (Figure 1C). Similar observations were made in the human amnion tissue-derived epithelial cell line WISH (Figure 1D), the human fibroblast cell-line GM637, and in PMA-differentiated monocyte/macrophage THP-1 cells (data not shown).
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Time course experiments revealed that the characteristic speckled distribution of IFP 35 was observed as early as 612 hr after IFN treatment and lasted for over 96 hr. Washing the cells after a 24-hr IFN treatment had no measurable effect on IFP 35 expression during the time period observed (96 hr; data not shown).
The subcellular distribution of IFP 35 was suggestive of vesicle-like structures. To identify the vesicular structure, IFN-treated HeLa cells were co-stained with antibodies specific for IFP 35 and antibodies targeting specific cell organelles: anti-mitochondrial antigen for mitochondria (
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A set of experiments was performed to investigate whether IFP 35 is associated with the cytoskeleton or the microtubule system. Staining with phalloidin to label actin filaments (
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Subcellular Localization
To determine the cellular compartment containing IFP 35, postnuclear supernatants from IFN-treated HeLa cells were fractionated on Percoll gradients. HeLa cells were treated with IFN- for 24 hr, homogenized, and cell debris and nuclei removed by centrifugation. The postnuclear supernatant was gradient-centrifuged in 15% Percoll (starting density 1.05 g/ml). To control for the integrity of cell organelles, Western blot analysis with antibodies against different compartments was carried out and demonstrated that the organelles were mostly intact (owing to the fragility of mitochondria, these organelles were partly disrupted by homogenization). The marker for early endosomes and plasma membrane (transferrin receptor) migrated in the low-density fractions (peak fraction 2), as did peroxisomes (peak fraction 2). The lysosomal marker cathepsin D and the mitochondrial marker peaked at densities higher than 1.065 g/ml. IFP 35 molecules were found in the least dense fractions (peak fraction 1 with trailing in the subsequent fractions), distinct from the endosome fractions and the peak of peroxisomes (see Figure 5A). Gradient centrifugation in 6.5% Percoll with a starting density of 1.04 g/ml for higher resolution of the low-density fractions separated IFP 35 from cellular organelles (see Figure 5B). IFP 35 peaked in the same fractions as ribosomes, demonstrating that this protein is confined to the cytoplasm. The results of the cell fractionation experiments are consistent with the apparent lack of co-localization of IFP 35 with organelles in the immunofluorescence studies, although they do not define the structures involved.
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To further characterize the cytoplasmic localization of IFP 35, gel filtration experiments to determine its native molecular mass were performed. Postnuclear supernatant from IFN-treated HeLa cells was separated by gel filtration using FPLC and a Superose 6 column (Figure 6). IFP 35, which by itself is a 35-kD protein (
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Electron microscopic studies were carried out to examine the structural basis for the immunofluorescence data. Electron microscopy applying the postembedding labeling technique using polyclonal anti-IFP 35 antibody and protein Agold complexes (10-nm gold particle size) demonstrated that IPF 35 did not localize to any known cellular compartment. Gold particles were mainly observed in clusters distributed throughout the cytoplasm. Clusters typically contained 612 gold particles, indicating that IFP 35 forms aggregates in the cytoplasm of IFN-treated cells (see Figure 7). Using Lowicryl K4M as embedding resin gave identical results (not shown). Counterstaining of the sections with uranyl acetate revealed that the clusters of IFP 35 were not associated with intracellular vesicles, the cell membrane, or the nuclear surface. In the statistical evaluation of the number of gold particles per cluster (over 50 clusters counted), only one cluster located at the cell membrane and two clusters in the vicinity of the nuclear surface were found. It is yet to be determined whether other proteins are involved in the formation of these structures, as suggested by the CHX experiments. Control cells not treated with interferon were almost devoid of any label; if at all, only single gold particles instead of clusters were found.
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Co-localization of Endogenous and Transfected IFP 35
Final proof for the described unique subcellular localization of IFP 35 was given by transfection studies. HeLa cells were transiently transfected with an expression vector coding for an IFP 35 protein tagged with the hemagglutinin (HA) epitope. As control, cells were transfected with an expression vector coding for HA-tagged B-ATF or with an insertless expression vector. Transfected cells were analyzed by Western blotting, immunofluorescence, and immunoprecipitation studies.
Owing to the insertion of the tag, the HA-tagged IFP 35 showed a slightly higher molecular mass than the endogenous IFP 35, allowing differentiation of endogenous and transfected IFP 35 by SDS-gel electrophoresis and Western blotting using IFP 35-specific antibodies. The HA-tagged IFP 35 partitioned in the same way as the endogenous IFP 35 protein, which is present predominantly in the nuclear fraction of cell extracts (
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Immunofluorescence studies of transfected cells with an anti-HA MAb showed the same speckled immunofluorescence pattern characteristic for endogenous IFP 35 (compare Figure 9 with Figure 1 and Figure 2). Double immunofluorescence studies using anti-HA MAb and polyclonal anti-IFP 35 antibody suggested co-localization of endogenous and transfected IFP 35 (not shown).
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To demonstrate complex formation between endogenous and transfected IFP 35, immunoprecipitation studies were performed. HA-tagged transfected proteins were immunoprecipitated with an anti-HA MAb and immunoprecipitates were subsequently analyzed by Western blotting using IFP 35-specific antibodies (see Figure 10). Co-immunoprecipitation of endogenous IFP 35 was observed in cells transfected with pDCR-IFP 35 but not in cells transfected with pDCR-B-ATF. As a control, aliquots of the cellular extracts used for immunoprecipitation were subjected to SDS-PAGE and submitted to immunoblotting using anti-HA and anti-IFP 35 antibodies. These controls demonstrated that quantitatively similar amounts of endogenous IFP 35 were present in the different cellular extracts investigated and that cells were efficiently transfected with pDCR-IFP 35 and pDCR-B-ATF.
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Discussion |
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IFP 35 is an IFN-regulated leucine zipper protein of unknown function, which forms homodimers in vitro. Previous studies have shown that IFP 35 was able to pellet in the nuclear fraction of crude cellular extracts (
The speckled pattern found by immunofluorescence microscopy was suggestive of vesicle-like structures. Co-localization experiments using double immunofluorescence failed to demonstrate an association with known organelles. Subcellular fractionation studies using Percoll gradient centrifugation also demonstrated a lack of association of IFP 35 with organelle-containing fractions. IFP 35 was found in the least dense fractions, indicative of a cytoplasmic localization. As a possible clue to the unique subcellular morphology of IFP 35, the native molecular mass of IFP 35 was determined by gel filtration of cytoplasmic extracts. These experiments demonstrated that IFP 35 eluted at a molecular mass of 200440 kD, suggesting that IFP 35 is part of larger protein complexes. Given that IFP 35 forms homodimers in vitro (
As a further proof to validate our observations on the subcellular localization of IFP 35, transfection experiments were carried out. In these experiments, an HA-tagged expression construct of IFP 35 was used. Staining of transfected cells with an HA-specific MAb showed the same speckled cytoplasmic expression pattern as observed with endogenous IFP 35 (compare Figure 9B with Figure 1 and Figure 2). As expected from the immunofluorescence results, separation of transfected cells into crude cytoplasmic and nuclear extracts demonstrated an identical partitioning for endogenous and transfected IFP 35 (see Figure 8). Immunoprecipitation of transfected cells using an HA-specific MAb and subsequent Western blot analysis of the immunoprecipitates demonstrated complex formation between endogenous and transfected IFP 35. This finding supports our previous observation that IFP 35 forms homodimers in vitro. Presumably, this interaction is at least in part mediated by the leucine zipper of IFP 35. The results from the immunoprecipitation experiments support the conclusion that the characteristic morphological structures of IFP 35, as observed by immunofluorescence and electron microscopy, consist of multimers of IFP 35 with or without other proteins. Our immunocytochemical and biochemical characterization strongly suggests that these structures represent large cytosolic protein aggregates. These structures are not associated with membrane-bound vesicles, as evident from subcellular fractionation and electron microscopic studies.
More recently, it has been suggested that IFP 35 forms complexes with B-ATF, a member of the AP1 family of nuclear, basic leucine zipper proteins (
An important question concerns the biological implications of our findings. Although no firm conclusions are yet possible in this respect, preliminary immuno-fluorescence experiments suggest a partial co-localization of IFP 35 with cytokeratins (M.B. Omary, personal communication; and our own unpublished results). The association of cytokeratins with proteins involved in signal transduction, such as the 14-3-3 protein (-related kinases (
There is precedence for binding of IFN-induced proteins to the cytoskeleton. The IFN-induced ubiquitin-crossreactive protein, a protein of 15 kD that has been suggested to target a set of proteins for degradation via the ubiquitin-dependent degradative pathway, has been shown to distribute in a cytoskeletal pattern (
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Acknowledgments |
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Supported in part by the Sonderforschungsbereich 244 "Chronische Entzündung" of the DFG. ECB is a Herman and Lilly Schilling professor.
We are indebted to B. Omary for communication of unpublished results and to K. v. Figura, B. Hoflack, J. Stahl, and J. Wehland for providing antibodies. We thank P. v. Wussow for gifts of interferon, M. Berger and I. Zellmann for excellent technical assistance, and two anonymous referees for helpful comments.
Received for publication April 23, 1998; accepted October 5, 1998.
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