The effect of arsenite-induced oxidative stress on the cellular location of RAP55. (A) In resting Hep-2 cells, RAP55-containing P-bodies were distributed throughout the cytoplasm, and (B) TIA was detected diffusely throughout the nucleus, but excluded from nucleoli. After 60 min of exposure to sodium arsenite, both (D) RAP55 and (E) TIA were detected in cytoplasmic granules. (H) After 1 h exposure to arsenite and 1 h recovery in culture medium, TIA-containing stress granules persisted in the cell cytoplasm. RAP55 was detected both within stress granules and in adjacent P-bodies (indicated by white arrows in G). To identify the portion of RAP55 that mediated localization to stress granules, GFP-RAP55 fragment fusion proteins were expressed in Hep-2 cells, which were then subjected to arsenite-induced stress for 1 h. (J) RAP55 amino acids 396–463 were necessary and sufficient to direct GFP to (K) TIA-containing stress granules. Overlap of green and red staining appears yellow in C, F, I, and L.
