FIGURE 3.

Nfs1p is activated by the addition of Isd11p. A, Nfs1p (100 ng) was incubated with PLP (150 μm) and [³⁵S]cysteine (5 μCi) for 10 min at 30 °C in the absence or presence of added Isd11p (100 ng). The preformed Nfs1p·Isd11p complex (100 ng) and Isd11p (100 ng) alone served as positive and negative controls, respectively. Proteins were precipitated with TCA, and samples were analyzed by non-reducing SDS-PAGE followed by autoradiography. DTT (10 mm) was added to the SDS sample buffer only as indicated (lane 5). B, increasing amounts of Isd11p were added to Nfs1p, and the reaction mixtures were supplemented with PLP (150 μm) and [³⁵S]cysteine (5 μCi). Following incubation at 30 °C for 10 min, proteins were precipitated, and samples were analyzed by non-reducing SDS-PAGE and autoradiography. C, Isd11p (100 ng) was added to Nfs1p (100 ng), and the reaction mixtures were supplemented with increasing concentrations of PLP. Following addition of [³⁵S]cysteine (5 μCi), samples were incubated for 10 min at 30 °C and subsequently analyzed. D, increasing amounts of Isd11p were added to Nfs1p (100 ng), and the reaction mixtures were supplemented with PLP (150 μm) and [³⁵S]cysteine (5 μCi). Samples were incubated for 10 min either on ice or at 25 °C and analyzed.