FIGURE 8.
Yfh1 or Isu1Sup does not promote exchange of Nfs1-bound substrate with free substrate. A, Nfs1 (100 ng) was supplemented with unlabeled cysteine (10 μm) and/or Yfh1 (100 ng) as indicated. All reaction mixtures contained PLP (0.15 mm). Samples were incubated at 30 °C for 15 min (first step). After ammonium sulfate precipitation, the protein pellets were solubilized with HS buffer plus 0.15 m NaCl, and Yfh1 (100 ng) was added to some samples as indicated. Following addition of [35S]cysteine (1× = 0.1 μm; 5 μCi), reaction mixtures were incubated at 30 °C for 10 min prior to addition of Isd11 (100 ng). The incubation at 30 °C was continued for another 15 min (second step). Following TCA precipitation, proteins were analyzed by nonreducing SDS-PAGE and autoradiography. B, same as in A except Yfh1 was replaced with Isu1Sup in both first and second steps of the assay.