Regulation of the Hap1 protein.

(A) HAP1-GFP is induced by iron starvation and by hemin. Cells expressing a HAP1-GFP allele from its native promoter (KC1368) were grown in regular YPD medium, or in YPD supplemented with 1 mM ferrozine or with 50 μM hemin for 4 hours, then visualized by epifluorescence microscopy. “No tag” indicates the untagged original CAI4 cells. (B) Hap1-GFP is located in the nucleus. The strain expressing Hap1-GFP under its native promoter, and a control untagged strain, were grown 4 h in YPD medium containing 1mM ferrozine and 10 μM hemin, then briefly stained with the DNA stain Hoechst 33342 and visualized by epifluorescence microscopy. (C) HAP1-GFP- expressing cells were grown 3 h in YPD supplemented with the indicated compounds at a uniform concentration of 50 μM, and visualized by epifluorescence microscopy. Top panels are GFP immunofluorescence micrographs, and bottom panels are immunofluorescence overlaid over DIC micrographs. (D) Hap1 is stabilized in the presence of hemin. Wild type cells carrying MAL2p-Hap1-6xMyc (KC1399), or a control strain (C = SN148) were grown overnight in YEP+ 2% raffinose at 30°C. In the morning, cells were diluted to OD600 = 0.5 in YEP+2% maltose, with or without 20 μM hemin, and incubated for 4 hours at 30°C. Next, 2% glucose was added, and the Hap1-6xMyc protein levels were followed by Western blotting. Time 0’ is immediately after glucose addition. Hap1-6xMyc was detected with the anti-Myc antibody 9E10, and the same membrane was reacted with an anti-tubulin antibody. Protein signals were visualized by chemoluminescence and quantitated. The left panel shows the membrane, and the right panel shows the ratio of Myc signal to tubulin signal, normalized to the signal at time 0’ for each culture (= 100%). The graph shows the average of two separate experiments. The error bars indicate standard deviations.