Functional analysis of the zinc cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells.
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.
Defranoux
N
N
Centre de Génétique Moléculaire du C.N.R.S., Laboratoire propre associé, Université Pierre et Marie Curie, Gif sur Yvette, France.
Gaisne
M
M
Verdière
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005809:Genes, Regulator; D006418:Heme / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors; D016335:Zinc Fingers / Q000235:genetics*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D006418:Heme