A cytoplasmic structure resembling large protein aggregates induced by interferons

IFP 35 is an interferon (IFN)-regulated leucine zipper protein, expression of which is observed in a variety of cell types including monocytes/macroph ages, epithelial cells and fibroblasts. Using immunofluorescence studies, w e demonstrate that IFP 35 is found in characteristic punctate cytoplasmic s tructures after IFN treatment. Co-localization experiments using double imm unofluorescence and confocal laser scanning microscopy failed to show assoc iation of IFP 35 with known organelles (mitochondria, peroxisomes, endoplas mic reticulum, lysosomes, endosomes, Golgi complex), ribosomes, or actin fi laments. Subcellular fractionation to separate membrane-associated from cyt oplasmic proteins demonstrated that IFP 35 localizes to the cytoplasm. Sepa ration of postnuclear supernatant from HeLa cells by gel filtration reveale d that IFP 35 eluted at a molecular mass of 200-440 kD, suggesting that IFP 35 is part of protein complexes. Electron microscopic studies showed cytop lasmic clusters of a few aggregates of IFP 35 in IFN-treated cells which we re neither associated with nor surrounded by a membrane. A combination of i mmunoprecipitation and immunofluorescence studies of cells transfected with a hemagglutinin epitope-tagged IFP 35 expression construct demonstrated co mplex formation and co-localization of endogenous and transfected IFP 35. T aken together, our studies demonstrate that IFP 35 associates with unique c ytoplasmic structures that are distinct from known organelles and resemble large protein aggregates.