METALLOREGULATION OF FRE1 AND FRE2 HOMOLOGS IN SACCHAROMYCES-CEREVISIAE

The high affinity uptake systems for iron and copper ions in Saccharom yces cerevisiae involve metal-specific permeases and two known cell su rface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2, Five novel genes found in the S. cerevisiae genome exhibit marked sequence simila rity to Fre1 and Fre2, suggesting that the homologs are part of a fami ly of proteins related to Fre1 and Fre2, The homologs are expressed ge nes in S. cerevisiae, and their expression is metalloregulated as is t rue with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifica lly hen-regulated through the Aft1 transcription factor. These genes a re expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake syst em. Expression of FRE3-FRE6 is elevated in AFT1-1(up) cells and attenu ated in aft1 null cells, showing that iron modulation occurs through t he Aft1 transcriptional activator. The fifth homolog FRE7 is specifica lly copper-metalloregulated, FRE7 is expressed in cells limited in cop per ion uptake by a Cu(I)-specific chelator or in cells lacking the hi gh affinity Cu(I) permeases, The constitutive expression of FRE7 in MA C1(up1) cells and the lack of expression in mac1-1 cells are consisten t with Mad being the critical transcriptional activator of FRE7 expres sion, The 5' promoter sequence of FRE7 contains three copper-responsiv e promoter elements. Two elements are critical for Mad-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expr ession. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are sepa rated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mad binding can be established a s TTTGC(T/G)C(A/G).