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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 1. Radiolabeling of a cytosolic component requires mitochondrial contribution of sulfur species.

Figure 1

(A) Reaction mixtures containing wild-type mitochondria (WT mito) alone, wild-type cytosol (WT cyto) alone, or both were supplemented with [35S]cysteine (10 μCi), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH and 1 mM NADPH), and ferrous ascorbate (10 μM). After incubation at 30°C for 30 min, samples were centrifuged, and the pellet (“P”; mitochondria) and supernatant (“S”; cytosol) fractions were analyzed by native PAGE and autoradiography. WT mito 1X = 50 μg of proteins; WT cyto = 300 μg of proteins.

(B) WT mitochondria (200 μg) alone were incubated with [35S]cysteine, nucleotides and iron. After centrifugation, the pellet and supernatant fractions were analyzed as in (A) above.

(C) Reaction mixtures containing different amounts of cytosol (1X = 50 μg) were supplemented with a fixed amount of mitochondria (200 μg), and incubated with [35S]cysteine, nucleotides and iron. Following centrifugation, samples were analyzed as in (A) above.