The yeast heme-responsive transcriptional activator Hap1 is a preexisting dimer in the absence of heme.
In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.
Hon
T
T
Department of Biochemistry, New York University Medical Center, New York, New York 10016, USA.
Hach
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A
Tamalis
D
D
Zhu
Y
Y
Zhang
L
L
D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006418:Heme / Q000378:metabolism* / Q000494:pharmacology; D018832:Molecular Chaperones / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D006418:Heme