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. Author manuscript; available in PMC: 2014 Jul 18.
Published in final edited form as: Cell. 2013 Jul 18;154(2):416–429. doi: 10.1016/j.cell.2013.06.043

Figure 4. Uba4p is negatively regulated during sulfur amino acid starvation.

Figure 4

(A) npr2Δ and npr3Δ strains exhibit excessive tRNA thiolation during sulfur starvation. Relative tRNA thiolation amounts were measured in WT, npr2Δ and npr3Δ cells grown in YPL or SL medium. Thiolation increased in npr2Δ and npr3Δ strains compared to WT cells only in SL (Mean±SD, n=3, with experimental duplicates. ** indicates p<0.01). See also Figure S4.

(B) Thiolation-deficient npr2Δ/uba4Δ strains exhibit slower growth compared to npr2Δ in SL medium. Growth curves of the indicated strains following switch from YPL to SL medium were measured over ~50 hours in batch cultures starting at an OD600 of ~0.1. (Mean±SD, n=3, *** indicates p<0.001). See also Figure S4

(C) npr2Δ cells have abnormally higher levels of sulfur-containing metabolites. Relative amounts of cysteine, methionine and SAM were measured in WT and npr2Δ cells growing in YPL or SL by LC-MS/MS

(D) Uba4p abundance decreases upon switch to SL medium. Western blot shows amounts of Myc-UBA4 present over time in cells growing in YPL, SL or SL+0.5mM methionine (Mean±SD, n=3)

(E) UBA4 and URM1 transcript levels remain relatively stable upon sulfur amino acid starvation. Relative amounts of UBA4 and URM1 transcripts in cells growing in the indicated conditions were measured by RT-qPCR (normalized to snR14). See also Figure S4.