Yeast lacking Stm1p are more sensitive to certain protein synthesis inhibitors. (A) Ten-fold serial dilutions of wild-type yeast strain K699 (wt) or its isogenic counterpart lacking Stm1p (stm1Δ) were plated on rich medium (YPD) containing 20 μg/ml anisomycin, as indicated. Colony growth after 3 days of incubation at 30°C is shown. (B) Same as in (A), except that yeast were plated on minimal synthetic defined medium supplemented with essential amino acids and nucleic acid bases (SD*) and containing 20 μg/ml anisomycin, 0.1 μg/ml cycloheximide, 50 μg/ml hygromycin B or 200 μg/ml paromomycin, as indicated.

Increased association of eEF3 with ribosomes in the absence of Stm1p. (A) Whole-cell extracts from wild-type K699 (wt) or its isogenic counterpart lacking Stm1p (stm1Δ) were resolved by ultracentrifugation through a 10–50% sucrose gradient and fractionated. Shown is an SDS–PAGE analysis of proteins present in the 80S fractions stained with Coomassie brilliant blue G-250. (B) UV absorbance trace from the sucrose gradient fractionation of wild-type K699 yeast extract (top) and western blots of ribosomal protein L3, eEF3 and Stm1p present in each fraction (bottom). (C) Same as in (B) except the cell extract was from stm1Δ yeast.

In the absence of Stm1p, eEF3 overexpression exacerbates the cell grow defect, reduces translational efficiency and increases eEF3 : ribosome association. (A) Ten-fold serial dilutions of wild-type yeast strain BY4741 (wt) or its isogenic counterpart lacking Stm1p (stm1Δ), transformed with either a galactose-inducible eEF3 expression plasmid (+eEF3) or a control plasmid (e), were plated on synthetic complete medium lacking uracil (SC-Ura), with either glucose (Glu) or galactose (Gal) and 20 μg/ml anisomycin (An), as indicated. Colony growth after 3 days of incubation at 30°C is shown. (B) Wt and stm1Δ BY4741 yeast, transformed with either a galactose-inducible eEF3 expression plasmid (+eEF3) or a control plasmid, were propagated in synthetic complete medium lacking uracil and containing galactose and 10 μCi [³⁵S]methionine for the durations indicated. Aliquots were removed and trichloroacetic-acid-precipitable radioactivity determined by scintillation counting. (C) UV absorbance trace from the sucrose gradient fractionation of whole-cell extract from wild-type BY4741 yeast transformed with an eEF3 expression plasmid (top, orange trace), stm1Δ BY4741 yeast transformed with a control plasmid (middle, cyan trace) and stm1Δ BY4741 yeast transformed with an eEF3 expression plasmid (bottom, red trace), each compared with the UV absorbance trace from the sucrose gradient fractionation of whole-cell extract from wild-type BY4741 yeast transformed with a control plasmid (blue traces). For each, their polysome to 80S ratio is indicated in brackets. All yeast strains were propagated for 4 h in SC-Ura medium plus galactose. (D) Proteins from whole-cell extracts from the yeast described in (C) were separated by SDS–PAGE and analyzed by western blotting using antibodies against Stm1p, eEF3 or actin, as indicated. In this experiment, actin served as a loading control. (E) Ribosomes from the aforementioned yeast were purified by sucrose gradient ultracentrifugation and proteins from sucrose gradient fractions corresponding to the 80S peak were separated by SDS–PAGE and analyzed by western blotting using antibodies against Stm1p, eEF3 or L3, as indicated. In this experiment, L3 protein served as a loading control.

Overexpression of Stm1p in a proteasome-deficient strain (AVL78) led to decreased amounts of eEF3 associated with ribosomes. (A) Ten-fold serial dilutions of wild-type yeast strain AVL78 transformed with either a galactose-inducible Stm1p expression plasmid (+Stm1p) or a control plasmid (e), were plated on synthetic complete medium lacking uracil (SC-Ura) and containing either glucose (Glu) or galactose (Gal), as indicated. Colony growth after 3 days of incubation at 30°C is shown. (B) AVL78 yeast transformed with either a galactose-inducible Stm1p expression plasmid (+Stm1p) or a control plasmid were propagated in synthetic complete medium lacking uracil and supplemented with galactose and 10 μCi [³⁵S]methionine for the durations indicated. Aliquots were removed and trichloroacetic-acid-precipitable radioactivity determined by scintillation counting. (C) UV absorbance traces from the sucrose gradient fractionation of whole-cell extracts from wild-type AVL78 yeast transformed with either an Stm1p expression plasmid (red trace) or a control plasmid (black trace). For each, their polysome to 80S ratio is indicated in brackets. All yeast strains were propagated for 4 h in SC-Ura medium plus galactose. (D) Proteins from whole-cell extracts of AVL78 were separated by SDS–PAGE and analyzed by western blotting using antibodies against Stm1p, eEF3 or actin, as indicated. (E) Stm1p from the above whole-cell extracts was immunoprecipitated with an anti-Stm1p antibody in the presence of 500 mM NaCl, proteins were resolved by SDS–PAGE and analyzed by western blotting using anti-ubiquitin antibodies (right). SDS–PAGE-resolved proteins from this experiment western blotted with anti-Stm1p antibodies are shown on the left. (F) Ribosomes from the above yeast extracts were purified by sucrose gradient ultracentrifugation and proteins from sucrose gradient fractions corresponding to the 80S peak were separated by SDS–PAGE and analyzed by western blotting using antibodies against Stm1p, eEF3 or L3, as indicated.

Role of Stm1p in translation elongation. Shown is a model of the yeast elongation cycle. Starting at the top left, Stm1p at high concentrations can inhibit the association of eEF3 (green ellipse) to post-translocation ribosomes. After ATP binding, which causes an elongated eEF3 conformation and unlocking of the E-site by moving the ribosome L1 stalk, bound Stm1p (red ellipse) can facilitate eEF3 dissociation from the ribosome and prevent reassociation. With eEF3 absent from the ribosome, the ternary complex eEF1A–GTP–aminoacyl-tRNA can then associate, delivering aminoacyl-tRNA (blue ellipse) to the ribosome A-site and releasing deacyl-tRNA (orange ellipse) from the E-site, steps inhibited by the antibiotic anisomycin (An). In the absence of Stm1p, eEF3 can inappropriately associate with ribosomes, thereby inhibiting subsequent ternary complex binding and deacyl-tRNA release and inhibiting efficient elongation, especially under conditions of translational stress (e.g. nutrient deprivation or antibiotic inhibition).