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Vol. 18, Issue 8, 2980-2990, August 2007

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Mechanism Underlying the Iron-dependent Nuclear Export of the Iron-responsive Transcription Factor Aft1p in Saccharomyces cerevisiae

Ryo Ueta^*,, Naoko Fujiwara^*, Kazuhiro Iwai^,, and Yuko Yamaguchi-Iwai^*,

*Department of Applied Molecular Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan; and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Saitama 322-0012, Japan

Submitted November 29, 2006; Revised May 15, 2007; Accepted May 18, 2007
Monitoring Editor: Karsten Weis

Aft1p is an iron-responsive transcriptional activator that plays a central role in maintaining iron homeostasis in Saccharomyces cerevisiae. Aft1p is regulated primarily by iron-induced shuttling of the protein between the nucleus and cytoplasm, but its nuclear import is not regulated by iron. Here, we have shown that the nuclear export of Aft1p is promoted in the presence of iron and that Msn5p is the nuclear export receptor (exportin) for Aft1p. Msn5p recognizes Aft1p in the iron-replete condition. Phosphorylation of S210 and S224 in Aft1p, which is not iron dependent, and the iron-induced intermolecular interaction of Aft1p are both essential for its recognition by Msn5p. Mutation of Cys291 of Aft1p to Phe, which causes Aft1p to be retained in the nucleus and results in constitutive activation of Aft1-target genes, disrupts the intermolecular interaction of Aft1p. Collectively, these results suggest that iron induces a conformational change in Aft1p, in which Aft1p Cys291 plays a critical role, and that, in turn, Aft1p is recognized by Msn5p and exported into the cytoplasm in an iron-dependent manner.

Address correspondence to: Yuko Yamaguchi-Iwai (yukoiwai{at}kais.kyoto-u.ac.jp) or Kazuhiro Iwai (kiwai{at}med.osaka-cu.ac.jp).

Abbreviations used: AD, Gal4 transactivation domain; BD, Gal4 DNA-binding domain; CIAP, calf intestine alkaline phosphatase; DAPI, 4',6'-diamino-2-phenylindole; FOB, ferrioxamine B; GFP, green fluorescence protein; HA, hemagglutinin; NES, nuclear export signal; NLS, nuclear localization signal; TAP, tandem affinity purification.