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. 2014 Nov;34(21):4062–4076. doi: 10.1128/MCB.00799-14

FIG 9.

FIG 9

Premature translation termination requires the translation termination factor Sup35. (A) Luc-K12-3HA was expressed in [PSI+] and [psi−] wild-type and Δzuo1 strains. Aliquots of total extracts were analyzed via immunoblotting using antibodies directed against luciferase (Luc) and Sse1. (B) Depletion of Sup35 is lethal. Δsup35 or Δzuo1 Δsup35 strains complemented with SUP35 on a Tet-off plasmid (wild-type-tet-SUP35 and Δzuo1-tet-SUP35, respectively) fail to grow in the presence of tetracycline (10 μg/ml). Tenfold serial dilutions of the indicated strains were spotted onto YPD plates with or without tetracycline. Growth was assessed after 3 days at 30°C. (C) Depletion of Sup35 for short periods of time leads to slow growth. Δzuo1-tet-SUP35 (see panel B) harboring GAL-Luc-K12-3HA was grown in glucose-containing liquid medium with or without tetracycline for the indicated times. Luc-K12-3HA expression was then induced by shifting cells to galactose-containing medium with tetracycline (+ tet) or without tetracycline (− tet). Growth was monitored after the galactose shift. The doubling times (td) are given. (D) Tetracycline treatment leads to Sup35 depletion. Δzuo1-tet-SUP35 (see panels B and C) was grown in the presence of tetracycline for the time periods indicated. The Δzuo1 strain served as a control for normal Sup35 levels. Total extracts were analyzed via immunoblotting using antibodies recognizing Sup35 and, as a loading control, Rpl24. (E) Sup35 is required for the expression of Luc-K12-3HA and Luc-K12t. The Δzuo1-tet-SUP35 strain harboring GAL-Luc-K12-3HA (see panel C) was grown on glucose-containing medium with or without tetracycline (tet) for the indicated periods of time and then shifted to galactose-containing medium with or without tetracycline for 7 h to induce Luc-K12-3HA expression. Aliquots of total extracts were analyzed as described above for panel D. (F) Sup35 depletion severely affects Luc-K12t expression. The Δzuo1-tet-SUP35 strain harboring GAL-Luc-K12-3HA (see panel C) was grown on glucose-containing medium with or without tetracycline for 20 h and then shifted to galactose-containing medium with or without tetracycline for the indicated periods of time. Aliquots of total extracts were analyzed as described above for panel D.