Schizosaccharomyces pombe cells acquire iron under high affinity conditions through the action of a cell surface ferric reductase encoded by the frp1 ؉ gene and a two-component iron-transporting complex encoded by the fip1 ؉ and fio1 ؉ genes. When cells are grown in the presence of iron, transcription of all three genes is blocked. A conserved regulatory element, 5-(A/ T)GATAA-3, located upstream of the frp1 ؉ , fip1 ؉ , and fio1 ؉ genes, is necessary for iron repression. We have cloned a novel gene, termed fep1 ؉ , which encodes an iron-sensing transcription factor. Binding studies reveal that the putative DNA binding domain of Fep1 expressed as a fusion protein in Escherichia coli specifically interacts with the 5-(A/T)GATAA-3 sequence in an iron-dependent manner. In a fep1⌬ mutant strain, the fio1 ؉ gene is highly expressed and is unregulated by iron. Furthermore, the fep1⌬ mutation increases activity of the cell surface iron reductase and renders cells hypersensitive to the iron-dependent free radical generator phleomycin. Mutations in the transcriptional corepressors tup11 ؉ and tup12 ؉ are phenocopies to fep1 ؉ . Indeed, strains with both tup11⌬ and tup12⌬ deletions fail to sense iron. This suggests that in the presence of iron and Fep1, the Tup11 and Tup12 proteins may act as co-repressors for down-regulation of genes encoding components of the reductive iron transport machinery.

Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

Alumina adsorption chromatography and ion-pair reversed-phase chromatography were developed to analyze the isomers of unprotonated and protonated n-butylamine Schiff base of retinal (RSB and PRSB), respectively. Photoisomerization starting from the all-trans, 11-cis and 13-cis isomers was traced for RSB in n-hexane, acetonitrile, methanol and 1-butanol, and for PRSB in methanol, acetonitrile and 1-butanol. The quantum yields of photoisomerization for the all-trans, 9-cis, 11-cis and 13-cis isomers were determined for RSB and PRSB in the above solvents except 1-butanol. On the other hand, photoisomerization of isomeric retinal bound (through Schiff base linkage) to bovine serum albumin (RBSA) in aqueous solution (pH 3, 7 and 12) as well as thermal isomerization of RSB (in n-hexane), PRSB (in methanol) and RBSA (in aqueous solution, pH 7) were traced starting from the all-trans, 11-cis, and 13-cis isomers. Protonation of RSB drastically changes the pathway of photoisomerization and increases the quantum yields of isomeric RSB. The solvent polarity increases the quantum yields of RSB differently depending on the configuration. Protonation enhances thermal isomerization also. The results of the above model systems are compared with those of retinal proteins to rationalize their selection of the particular isomerization pathways.

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Thirty‐two protein phosphatase (PPase) genes were identified in the genome nucleotide sequence of Saccharomyces cerevisiae. We constructed S. cerevisiae disruptants for each of the PPase genes and examined their growth under various conditions. The disruptants of six putative PPase genes, i.e. of YBR125c, YCR079w, YIL113w, YJR110w, YNR022c and YOR090c, were created for the first time in this study. The glc7, sit4 and cdc14 disruptants were lethal in our strain background. The remaining 29 PPase gene disruptants were viable at 30°C and 37°C, but only one disruptant, yvh1, showed intrinsic cold‐sensitive growth at 13°C. Transcription of the YVH1 gene was induced at 13°C, consistent with an idea that Yvh1p has a specific role for growth at a low temperature. The viable disruptants grew normally on nutrient medium containing sucrose, galactose, maltose or glycerol as carbon sources. The ppz1 disruptant was tolerant to NaCl and LiCl, while the cmp2 disruptant was sensitive to these salts, as reported previously, and none of the other viable PPase disruptants exhibited the salt sensitivity. When the viable disruptants were tested for sensitivity to drugs, i.e. benomyl, caffeine and hydroxyurea, ppz1 and ycr079w disruptants exhibited sensitivity to caffeine. Copyright © 1999 John Wiley & Sons, Ltd.

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The Schizosaccharomyces pombe fep1 ؉ gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11 ؉ and tup12 ؉ genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11 ؉ -and tup12 ؉ -encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted ␤-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.

We have cloned a DNA fragment complementing the aarl mutation defective in the al-a2 repression of the al cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR] gene is identical with TUPI, which is allelic to the SF12, FLKI, CYC9, UMR7, AMMI, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR] gene in a and a/a cells conferred the nonmating phenotype, and the a/a diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for al-a2 repression of RMEI and for a2 repression of a-specific genes.