The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3—lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt −435 to −350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt −348 to −247 that do not contain obvious Aft1 binding sites. The −348 to −247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.
Abbreviations used: BPS, bathophenanthroline-disulphonic acid; DMT1, divalent metal transporter; HA, haemagglutinin; MDM, metal-depleted minimal defined medium; SD, synthetic dextrose; SD/LF, SD/low iron; UAS, upstream activating sequence.
Present address: Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, U.S.A.
These authors contributed equally to this work.