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. Author manuscript; available in PMC: 2019 Jun 21.
Published in final edited form as: Cell Chem Biol. 2018 Apr 26;25(6):738–748.e3. doi: 10.1016/j.chembiol.2018.04.002

Figure 3. Nfs1 and Isu1, but not Ssq1, are required for cytosolic tRNA thiolation.

Figure 3

(A) WT cytosol (200 μg) was supplemented with WT or nfs1-14 mitochondria (100 μg). After addition of [35S]cysteine, nucleotides and iron, samples were incubated at 30°C and centrifuged. The resulting pellet (“P”, mitochondria) and supernatant (“S”, cytosol) fractions were analyzed as in Figure 1A legend.

(B) Isu1-depleted (Isu1↓; Isu2 absent) or WT mitochondria (250 μg) were added to WT cytosol (200 μg), and reactions mixtures were incubated with [35S]cysteine, nucleotides and iron at 30°C. After centrifugation, the pellet (mitochondria) and supernatant (cytosol) fractions were analyzed as in Figure 1A legend.

(C) WT or ssq1 mutant mitochondria (250 μg) were added to WT cytosol (200 μg), and assays were performed as in (B) above. See also Figure S1.