FIGURE 7.

Abc3 is a hemin-binding protein. A, hem1Δ shu1Δ abc3Δ cells expressing HA₄-tagged Shu1 and GFP-tagged Abc3 were precultured in the presence of ALA (200 μm) and Dip (50 μm). After washing, cells were transferred to ALA-free medium. At this point, they were treated with Dip (250 μm) or FeCl₃ (100 μm) for 6 h and then whole cell extracts were prepared. Cell membranes were obtained by ultracentrifugation of whole cell extracts and treated with Triton X-100 to release membrane proteins. Triton X-100-solubilized membrane proteins (input) were subjected to hemin pulldown assays using hemin-agarose or agarose alone. Unbound and bound protein fractions were analyzed by immunoblot assays using anti-GFP, anti-HA, and anti-PCNA antibodies. Soluble fractions contain proteins found in the supernatant after ultracentrifugation of whole cell extract preparations. The positions of molecular weight standards are indicated on the left. Abc3-GFP, Shu1-HA₄, and PCNA are indicated with arrows.