Functional analysis of the zinc cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells

Abstract

CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under heme-deficient conditions is dependent upon DNA binding.

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

“…They include S. cerevisiae Ume6p, as well as C. albicans Czf1p (283). Several mutagenic studies demonstrate the importance of the six cysteine residues in DNA binding and protein function (12,50,79,111,204,205,248,260,293). Other residues found within the metal-binding motif are equally important.…”

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase

“…The yeast chromosome blot was obtained from Clontech and hybridized according to the manufacturer's instructions. Total RNA was purified according to Defranoux et al (1994), and the Northern blots were hybridized using the Clontech protocol with end-labelled oligonucleotide probes specific for each gene: CIT1 5Ј-CTTTTGCTAGTTGTTGATAATATCGCTG-AC-3Ј; CIT2, 5Ј-CACGAAATCTTCATCCTTAGAACCAATC-AAGTTG-3Ј; and CIT3, 5Ј-CATGCCGAGCAGGGAACATAT-GTTTGTACACC-3Ј.…”

Iron Regulation through the Back Door: Iron-Dependent Metabolite Levels Contribute to Transcriptional Adaptation to Iron Deprivation in Saccharomyces cerevisiae

“…This tandem structure is shared by other genes involved in respiration (40,45). While the activating molecule of the Hap2-5 complex is unknown, Hap1 requires heme for transcriptional activity (6,55,56). In order to quantify the contribution of the CYC1 terminator to the iron-responsive expression of CYC1, we employed a luciferase reporter construct, p⌬UAS1/2-hRluc, which lacks the two upstream activating domains (Fig.…”

“…The above data show that the iron-dependent remodeling of levels of a key regulatory molecule of a biochemical pathway confers an iron-responsive gene expression pattern. In S. cerevisiae, the iron-containing cofactor heme functions as a key regulatory metabolite during the adaptation to hypoxic conditions (6,55,56). Since heme levels are low in iron-deprived cells, it was tempting to speculate whether heme also functions as a regulatory molecule in iron-responsive expression of genes involved in respiration.…”

“…Thus, similarly to LEU1, a reduced transcriptional activation of CYC1 goes along with a diminished iron responsiveness. We then studied the role of Hap1 in iron-responsive transcription of CYC1 which binds to the UAS1 region (6,18,55,56). Most laboratory yeast strains harbor a transposon insertion at the HAP1 locus that replaces the C-terminal 12 amino acids with a 30-amino-acid random sequence (14).…”