FINAL ACCEPTED VERSION: C-00314-2002.R1 Mammalian Stress Granules Represent Sites of Accumulation of Stalled Translation Initiation Complexes

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNAi, (Met-tRNAi) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2•GTP•Met-tRNAi complex is thought to bind to the 40S ribosomal subunit before the eIF4G•eIF4E•mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2, but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the a-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.