Molecular Mechanism Governing Heme Signaling in Yeast: a Higher-Order Complex Mediates Heme Regulation of the Transcriptional Activator HAP1

Abstract

Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization. To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast hemeresponsive transcriptional activator HAP1. HAP1 activity is regulated precisely and tightly by heme. Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex. Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1. The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity. Further, we determined HAP1 domains required for heme regulation: three domains-the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain-cooperate to form the higher-order complex and mediate heme regulation. Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation. Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…Studies have revealed that Hap1 contains several important functional elements and that heme regulation of Hap1 involves the regulation of DNA-binding and transcriptionactivating activities of Hap1 (Figure 2A) [14,[28][29][30][31]. The C6 zinc cluster motif, which is located near the N-terminus, and the dimerization domain mediate Hap1 DNA binding, while the seven heme-responsive motifs (HRMs) and three repression modules (RPMs) are responsible for coupling heme regulation with Hap1 activation (Figure 2, Table 1) [14,29,[31][32][33][34].…”

Regulation of the HAP1 gene involves positive actions of histone deacetylases

“…Yeast strains used were JEL1 (MAT α leu2 trp1 ura3-52 nprb1-1122 pep4-3 ΔHis3::pGAL10-GAL1) [26], BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), BY4741Δsnf2::Mxkan4, BY4741Δsin3::Mxkan4, BY4741Δrpd3::Mxkan4, BY4741Δhda1::Mxkan4, BY4741Δhos1::Mxkan4, BY4741Δ hos2::Mxkan4, BY4741Δ hos3::Mxkan4. The BY4741 strains [27] were purchased from Open Biosystems.…”

“…Yeast JEL1 (MATα leu2 trp1 ura3-52 nprb1-1122 pep4-3ΔHis3::pGAL10-GAL1) [26] cells were transformed with the Hap1 expression plasmid under the control of the GAL1 promoter or its own promoter [26]. Cell extracts were prepared as described previously [26].…”

“…Cell extracts were prepared as described previously [26]. Protein concentrations were determined by the BCA (bicinchoninic acid) protein assay kit (Pierce).…”

“…Electrophoretic mobility shift assays (EMSAs) and DNA pull down DNA-binding reactions were carried out exactly as described [17,26,28]. Radioactivity of the interested bands was visualized and quantified by using the PhosphoImage ™ system (Molecular Dynamics).…”