FIG. 1.
Effect of prt1-rnp1 on cell growth and translation initiation in vivo. (A) Schematic representation of PRT1, showing the location of prt1-1, the RRM domain, and the RNP1 and RNP2 motifs. Below is a sequence alignment of this region in yeast (S.c., Saccharomyces cerevisiae) PRT1 and residues in RRM2 of human (H.s., Homo sapiens) PABP, together with the seven residues that are changed to alanines in prt1-rnp1. Letters in boldface indicate amino acids in PABP RRM2 that make direct contact with RNA (9). (B, left) Growth curves of isogenic PRT1 (H2879), prt1-rnp1 (H3674), and prt1-1 (H1676) cells in liquid yeast extract-peptone-dextrose (YPD) medium at 25°C. At the time indicated by the arrow, half of each culture was harvested and resuspended in YPD at 37°C and cultured for the indicated time (B, right). (C) prt1-rnp1 strain H3674 was transformed with low-copy (lc) PRT1 plasmid p2625 or vector pRS316, and WT PRT1 strain H2879 was transformed with pRS316. Tenfold serial dilutions of the cells were spotted on solid SC lacking uracil (SC-Ura) and incubated at 25 or 34°C. (D) Western analysis of WCEs from PRT1 (H2879) and prt1-rnp1 (H3674) cells grown in YPD at 25°C and treated for 1 h at 37°C with antibodies to PRT1 and TIF11/eIF1A. Signals were quantified by using NIH image software, PRT1/eIF1A ratios were calculated, and both ratios were normalized to that calculated for the PRT1 strain, with the final value shown below the PRT1 blots. (E) PRT1 (H2879) and prt1-rnp1 (H3674) cells were grown in YPD at 25°C, treated for 1 h at 37°C, and cross-linked with 1% HCHO prior to harvesting. Fifteen optical density units at 260 nm of WCE were separated by velocity sedimentation through a 4.5 to 45% sucrose gradient, and the fractions were collected starting from the top of the gradient while scanning at A254 to visualize ribosomal species and polysomes, as indicated. (F) Same as in panel E except that cells were not heat treated. Doubling times of the strains (Td) at 25°C are indicated.