FIGURE 8.

In vitro and in vivo functional analysis of Dph4. A, in vivo rescue of Δjjj3 phenotype by DPH4. All strains indicated contain DT plasmid under galactose regulation. Serial dilutions of wild type strain with empty vector (W303), Δjjj3 with empty vector (−), or 2μ-GPD vector expressing full-length JJJ3 (Jjj3) or full-length DPH4 (Dph4) were spotted on dropout media containing 2% glucose or galactose and incubated for 72 h at 30 °C. B, UV-visible absorption spectra of 200 μm Fe-Dph4 and iron-bound Jjj3 (Fe-Jjj3) purified from E. coli grown in iron-supplemented M9 medium recorded at 25 °C. Inset, nickel bead-bound Fe-Dph4 and iron-bound Jjj3 proteins. C, iron-bound Jjj3 separated on native-PAGE and stained for iron. D, immunodetection of iron-bound Jjj3 by anti-His antibody. Oligomers are marked with arrows.