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. 2014 Aug 21;42(16):10321–10330. doi: 10.1093/nar/gku670

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — 5MPs from diverse eukaryotes bind and inhibit eIF2 in yeast. (A) Expression in yeast S. cerevisiae. Indicated amounts of whole cell extracts (WCEs) prepared from KAY33 transformants carrying an empty vector (Vec), or pEMBL-based plasmid coding for Tca, Tae, Ptr and Gin 5MP and grown in SCGal-Ura medium were subjected for immunoblotting with antibodies listed to the left. Asterisks show cross-reactivity to unknown yeast proteins. (B) Affinity purification. Complexes containing the FLAG-tagged (FL-) 5MPs were purified from KAY33 transformants carrying the plasmids used in (A) as described (15). Note that 5 μl (panel labeled FLAG) or 20 μl (all other panels) of the eluates were analyzed by Coomassie Blue staining and immunoblotting with antibodies raised listed to the right. (C) Graph summarizing the relative amounts of eIF2 or eIF3 associated in vivo with FL-5MP, compared to their total amounts in yeast. These values were computed based on molar ratios of eIF2, eIF3 and FL-5MP in the purified fractions, the extent of FL-5MP overexpression compared to FLAG-eIF5 and known stoichiometry of eIF2, eIF3 and eIF5 in vivo (18). (D) Model for 3AT resistance by overexpression of eIF5 or 5MP in gcn2Δ strain. Dashed stop bar indicates a weak inhibition. (E) Yeast growth assays. Transformants of strain KAY33 (gcn2Δ GCD6) (row 1) and KAY34 (gcn2Δ gcd6–7A) (rows 2–9) carrying an empty vector (Vec in rows 1, 2, 7), YEpU-TIF5 (Sce eIF5 in row 3), pEMBL-5MP derivatives expressing 5MP from indicated species were grown in SC-His-Ura medium. Fixed amounts (A₆₀₀ = 0.15) of the culture and its 10-fold serial dilutions were spotted onto the agar plates of SCGal-Ura medium lacking histidine without (panel 1) or with 60 mM 3AT (panel 2) and incubated at 30ºC for 3 and 8 days, respectively. (F) Expression from chromosomally integrated HIS4-lacZ in KAY34 (gcd6–7A) transformants used in panel E or one carrying pEMBL-FL-5MP1 (5) (Hsa 5MP1) and grown in SCGal-Ura medium for 6 h was presented by relative β-galactosidase units compared to vector control (as 100%). The β-galactosidase units expressed from HIS4-lacZ in vector controls are 114.8 and 73.8, respectively, for panels 1 and 2. Asterisk, the P-value for the difference compared to vector control is <0.05.