Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1.

Heme plays key roles in oxygen sensing and utilization in all living organisms ranging from bacteria to humans. In mammals, recent experiments suggest that hemoproteins are the oxygen sensors in the induction of the synthesis of erythropoietin and vascular endothelial cell growth factor in response to hypoxia (13, 22, 25). Heme also directly regulates various molecular and cellular processes for systems that use or sense oxygen. For example, heme stimulates the expression of globin chains in erythroid cells and cytochrome P-450 in hepatic cells (6, 9, 36), activates protein synthesis through the heme-regulated inhibitor kinase (7), and regulates the transport of numerous enzymes and the assembly of hemoglobin and cytochrome complexes (24, 31). The roles of heme are largely conserved from yeast to humans, and recent evidence indicates that heme might regulate diverse processes through similar mechanisms (24, 48). For example, recent experiments (32, 37) suggest that heme regulation of gene transcription in mammalian cells is mediated by heme-responsive transcription factors like the yeast transcriptional activator HAP1 (8, 34). However, no mammalian heme-responsive transcriptional regulator has been identified to date. Nonetheless, the yeast HAP1-heme regulatory system provides an excellent model for studying heme signaling in eukaryotic cells. In the yeast Saccharomyces cerevisiae, heme functions as the internal barometer of oxygen tension; heme synthesis in mitochondria is directly correlated with oxygen levels in the extracellular environment (23, 29). Heme induces the expression of many genes encoding respiratory functions and functions involved in controlling oxidative damage (15, 19, 50). The effect of heme on gene expression is mediated by the transcriptional activator HAP1 (8, 34), which is the only known eukaryotic heme-responsive transcriptional regulator. HAP1 activity is precisely controlled by heme concentrations: HAP1 activity gradually increases as the heme concentration increases and reaches the limit at micromolar heme concentrations (46). Presumably, heme binds to HAP1 and activates HAP1, which in turn promotes transcription from many promoters (8, 34). At low levels of HAP1 expression (as occurs naturally from its own promoter), HAP1 activity can be induced 1,000-fold by heme, while at high levels of expression (from the inducible GAL1-10 promoter), HAP1 activity is induced approximately 50-fold by heme (15, 46, 47). HAP1 contains five functional domains (33, 44, 48, 49): the C6 zinc cluster, the dimerization domain, the heme domain, the heme-responsive motif 7 (HRM7) domain, and the activation domain. The heme domain and the HRM7 domain contain six HRMs and one HRM, respectively; both domains have been shown to be involved in heme regulation of HAP1 (8, 33, 48). HAP1 DNA binding requires the C6 zinc cluster and the dimerization domain. The C6 zinc cluster is a highly conserved DNA recognition motif found in at least 40 yeast transcriptional activators including GAL4 and PPR1 (12, 27, 28, 38, 39). The HAP1 dimerization domain also contains a coiled-coil dimerization element homologous to that within the dimerization domain of GAL4 or PPR1 (27, 38, 44). HAP1, however, is a unique member of the C6 zinc cluster family: it recognizes asymmetric DNA sites and binds to DNA asymmetrically, whereas GAL4 and PPR1 recognize symmetric DNA sites and bind to DNA symmetrically (27, 28, 39, 49). More intriguingly, how does heme regulate HAP1 activity? Previous results suggest that heme stimulates HAP1 DNA-binding and transcriptional activity through both the heme domain and the HRM7 domain (33, 48). In addition, it has been shown that in the absence of heme, HAP1 appears as a slowly migrating band when analyzed by DNA mobility shift assays (11, 47). This band was designated a high-molecular-weight complex (HMC) (47). Heme disrupts this putative HMC and permits HAP1 to bind with high affinity to DNA as a dimer (47). This observation raises many questions. For example, is the HMC really a large and biochemically distinctive complex containing multiple proteins? Or is it an artifact of DNA mobility shift assays? Or is it just an aggregate of HAP1 formed in the absence of heme? Further, if the HMC is indeed a biochemically distinctive complex, what is its functional role in mediating heme regulation of HAP1? What HAP1 elements are required for the formation of the HMC and for mediating heme regulation? To answer these questions and to test the hypothesis that the HMC represses HAP1 activity in the absence of heme, we carried out a series of experiments. Using sucrose gradient centrifugation and affinity and gel filtration chromatography, we show here that the HMC is a biochemically distinctive complex and contains HAP1 and four other cellular proteins including heat shock proteins Hsp82 and Ydj1. We further show that the formation of the HMC is directly correlated with HAP1 heme responsiveness. Our data strongly suggest that the HMC is responsible for heme regulation. We also explored the possibility that the HAP1 dimerization domain is important for heme regulation and transcriptional activation. Strikingly, domain-swapping experiments show that the HAP1 dimerization domain is critical for heme regulation and transcriptional activation. We demonstrate that three domains—the heme domain, the HRM7 domain, and the dimerization domain—are all required for the formation of intact HMC and heme regulation of HAP1 activity.