Fig. 7. Kra is a translation inhibitor. (A) Kra cosediments with the 40S ribosomal subunit in a sucrose gradient of S2 cell extracts. Cell extracts were prepared in the presence of 100 µg/ml cycloheximide and fractionated on a 7-47% sucrose gradient. Fractions were analyzed for absorbance at 260 nm and subjected to SDS-PAGE and western blot analysis using anti-Kra, anti-eIF4E and anti-L28 antibodies. Positions of 40S and 60S ribosomal subunits, 80S ribosomes, and polysomes are indicated. (B) Kra binds eIF2ß in vitro. An SDS-gel stained with Coomassie Blue shows GST, GST-eIF5 and GST-Kra proteins in the amounts used for pull-down assays (top panel). A recombinant GST-Kra fusion protein, but not GST alone, interacts with [³⁵S]methionine-labeled eIF2ß (bottom panel). GST-eIF5 was used as a positive control for binding. (C) The AA-boxes in the W2 domain of Kra are important for its efficient binding to eIF2ß in vivo. HA-tagged Kra-WT, -12A, or -7A was coexpressed with GFP-tagged eIF2ß in the Drosophila embryo. Embryo extracts were immunoprecipitated with anti-HA, and the precipitated eIF2ß was detected by western blot analysis using anti-GFP. (D) Shot and eIF2ß can simultaneously bind to Kra. Soluble extracts of S2 cells overexpressing C-Shot L-GFP were immunoprecipitated with anti-GFP or IgG, and the presence of endogenous Kra and eIF2ß in the precipitates was determined by western blot analysis using either anti-Kra or anti-eIF2ß antibody, respectively. (E) Kra inhibits translation in vitro. In the top panel, luciferase mRNA was translated in reticulocyte lysates with [³⁵S]methionine in the absence and presence of the indicated proteins (2.4 µM); reaction samples were analyzed by SDS-PAGE and autoradiography. Translation of luciferase mRNA is specifically inhibited by the addition of Kra. In the middle panel, 10% of each reaction was removed after incubation and analyzed by RT-PCR for the presence of luciferase mRNA. In the bottom panel, the indicated amounts of GST, Kra-WT and Kra-7A were added to reticulocyte lysates. Cyc, cycloheximide (100 µg/ml).