To identify signaling proteins mediating UPR-induced cell destruction, we purified poly-ribosomes to enrich for mRNAs that become preferentially translated (Heiman et al., 2008) very early in response to catastrophic ER stress. A gene encoding an enhanced green fluorescent protein (EGFP) epitope target was fused to the large ribosomal subunit protein L10a, and expressed in INS-1 insulinoma cells, which are differentiated insulin-producing cells derived from rat pancreatic islets (Figure 1A). The chimeric gene (or an EGFP control) was driven from a tetracycline-inducible expression construct integrated at a chromosomal FRT docking site in the INS-1 cells. Exposed to doxycycline (Dox), the cells express the EGFP-L10a fusion, or EGFP (Figure 1B). Compared to EGFP, which localizes primarily to the cytosol, EGFP-L10a localizes to both cytosol and nucleosomes, consistent with assembly into ribosomes (Figure 1C) (Heiman et al., 2008).
