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. 2019 Jul 12;47(16):8807–8820. doi: 10.1093/nar/gkz600

Figure 1.

Figure 1.

The essential translational factor eEF3 is dispensable upon overexpression of New1. (A) The domain structure of Saccharomyces cerevisiae and Schizosaccharomyces pombe New1 as well as S. cerevisiae eEF3. The location of the catalytic glutamate residues essential for ATPase function is indicated. (B) Loss of NEW1 increases the growth defect caused by depletion of eEF3. The wild-type (VKY9), PMET25-YEF3 (VKY8), new1Δ (MJY945) and PMET25-YEF3 new1Δ (MJY951) strains were grown overnight in liquid SC-met-cys medium, 10-fold serially diluted, spotted on SC-met-cys (0 mM met) and SC-cys (0.5 mM met) plates. The plates were scored after three days incubation at 30°C. (C) Increased dosage of NEW1 counteracts the growth defect caused by reduced eEF3 levels. The PMET25-YEF3 strain carrying the indicated low-copy (l.c.) or high-copy (h.c.) URA3 plasmids was grown overnight in liquid SC-ura-met-cys medium, 10-fold serially diluted, spotted on SC-ura-met-cys (0 mM met) and SC-ura-cys (2 mM met) plates, and incubated at 30°C for 3 days. (D) Increased expression of NEW1 counteracts the inviability of cells lacking eEF3. The yef3Δ strain harbouring the l.c. URA3 plasmid pRS316-YEF3 (VKY20) was transformed with the indicated l.c. or h.c. LEU2 plasmids. The transformants were grown over-night in liquid SC-leu medium, 10-fold serially diluted, spotted onto SC-leu and SC-leu+5-FOA plates, and incubated at 30°C for three (SC-leu) or 5 (SC-leu+5-FOA) days. On 5-FOA containing plates only those cells that have lost the URA3 plasmid are able to grow (74). (E) Increased dosage of the YEF3 gene does not suppress the growth defect of new1Δ cells. The cells harbouring the indicated plasmids were grown overnight in liquid SC-ura medium, 10-fold serially diluted, spotted on SC-ura plates and incubated at 30°C for 2 or at 20°C for 4 days.