Fig. 1.
Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1. b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species.70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d. Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional PGAL1-RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in PGAL1-RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies