Fig. 2: Reh1 + 6 blocks Efl1 function.
From: The assembly factor Reh1 is released from the ribosome during its initial round of translation
a Reh1 + 6 inhibits the GTPase activity of Efl1. Efl1 GTPase activity was monitored by the production of GDP in reactions containing the indicated combinations of 80 nM 60S subunits, 100 nM Sdo1, 60 nM Efl1 and 80 nM (1x relative to 60S) or 400 nM (5x relative to 60S) Reh1 (WT or Reh1 + 6). For reactions containing 60S, Efl1, Sdo1 and Reh1, the nonspecific GTPase activity contributed by the Reh1 preps was subtracted. Mean and standard deviation are shown. All experiments were done in triplicate. Two-tailed t-test; P value = 0.0443. b Fluorescence microscopy of Tif6-GFP expressing cells (AJY4049) containing vector or expressing REH1 WT (pAJ4751) or REH1 + 6 (pAJ4157). DAPI staining for nuclei and differential interference contrast (DIC) images are shown for reference. c Similar to panel (a) except strain AJY4050, expressing Nmd3-L505A-GFP, was used. d Reh1 + 6 does not block recycling of the upstream factors Nog1, Mrt4 and Arx1. Similar to panel (a) except that strains were AJY4659 (Nog1-GFP), AJY4661 (Arx1-GFP) and AJY4660 (Mrt4-GFP) and only DIC is shown for reference. e, The dominant negative effect of REH1 + 6 is suppressed by TIF6-V192F. Ten-fold serial dilutions of wild-type yeast (BY4741) containing vectors expressing the indicated factors were plated on galactose-containing medium. Vectors used were: REH1 (WT) pAJ4751, REH1 + 6 (pAJ4157), vector (pRS415), TIF6 (WT) pAJ2846, TIF6-V192F pAJ2249. f Reh1 + 6 enters polysomes when suppressed by TIF6-V192F. Extracts of TIF6-V192F mutant cells (AJY4654) expressing REH1 WT (pAJ4751) or REH1 + 6 (pAJ4157) were separated by sucrose density gradient sedimentation. UV trace monitoring rRNA is shown. Fractions were analyzed by western blotting for the presence of Reh1 and Rpl8. The microscopy experiments shown in panels b, c and d are representative cells from fields of more than 100 cells from single micrographs. The growth assay shown in panel e is representative of multiple isolates of each transformed strain. The experiments in panel f are representative of two independent experiments. Source Data are provided as a Source Data file.
