FIG. 3.

40S binding of eIF3 core subunits is reduced in prt1-rnp1 cells at the nonpermissive temperature. (A) PRT1 (H2879) or prt1-rnp1 (H3674) cells were grown in YPD at 25°C, heated for 30 min at 37°C, and cross-linked with 2% HCHO prior to harvesting. Twenty optical density units at 260 nm of WCE were separated by velocity sedimentation on a 7.5 to 30% sucrose gradient, and 0.7-ml fractions, numbered from the top of the gradient (7.5%), were divided into 0.2- and 0.5-ml aliquots and analyzed by Western and Northern analysis, respectively, to detect eIFs, 40S subunit protein RPS22, RPL41A mRNA, and . The two to three fractions containing 40S subunits and 43S/48S preinitiation complexes are labeled above the Western blot as “40S.” Aliquots of the starting WCEs were analyzed in parallel (Input). (B) Amounts of each factor and RNA in the 40S fractions were quantified by phosphorimaging or fluorescence imaging analyses from five replicate experiments, and results for the rnp1 mutant were normalized to the corresponding PRT1 values and averaged. The data for the five eIF3 core subunits were combined and averaged (eIF3). The means and standard errors were plotted in the histogram. (C) Same as in panel A except for no heat treatment.