FIGURE 4.

Nfs1p binds cysteine in the absence of Isd11p but can utilize bound cysteine for persulfide formation only in the presence of Isd11p. A, Nfs1p (100–200 ng) was incubated with PLP (150 μm) and [³⁵S]cysteine (5 μCi) in a final volume of 50 μl for 10 min at 30 °C. Following addition of saturated ammonium sulfate (100 μl), samples were incubated for 1 h on ice and centrifuged at 15,000 × g for 30 min. The protein precipitates were solubilized with buffer A, supplemented with PLP (150 μm), and then incubated at 30 °C for 10 min with or without added Isd11p (100–200 ng). Proteins were again precipitated with TCA and analyzed by non-reducing SDS-PAGE followed by autoradiography. B, Nfs1p (200 ng) was incubated with [³⁵S]cysteine (5 μCi) in the absence or presence of PLP (150 μm) for 10 min at 30 °C. Following ammonium sulfate precipitation, samples were supplemented with Isd11p (200 ng), incubated at 30 °C for 10 min with or without PLP (150 μm), and analyzed as in A. C, a mixture of PLP (150 μm) and [³⁵S]cysteine (5 μCi) was incubated without or with different amounts of Nfs1p at 30 °C for 10 min. Following addition of the preformed Nfs1p·Isd11p complex (100 ng) as indicated, samples were further incubated at 30 °C for 10 min. Proteins were precipitated with TCA, and samples were analyzed as in A.