A New Class of Repression Modules Is Critical for Heme Regulation of the Yeast Transcriptional Activator Hap1

Abstract

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

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

“…HRMs are short sequence motifs that contain Cys residues [14,21,29,38,39] (Figure 2B). Not all of the seven HRMs are equally important for the activation of Hap1 [40,41]. Experimental evidence suggests that HRM7 is critical for heme activation of Hap1, while HRMs 1-6 appear to play auxiliary roles and are dispensable for Hap1 activation by heme [40,41].…”

“…Not all of the seven HRMs are equally important for the activation of Hap1 [40,41]. Experimental evidence suggests that HRM7 is critical for heme activation of Hap1, while HRMs 1-6 appear to play auxiliary roles and are dispensable for Hap1 activation by heme [40,41]. Heme promotes Hap1 transcriptional activity by enhancing the association of Hsp90 to the Hap1-Hsp70-Ydj1-Sro9 multichaperone complex, causing conformational changes in the complex [14,29,31,34,36,37,40,41].…”

“…Experimental evidence suggests that HRM7 is critical for heme activation of Hap1, while HRMs 1-6 appear to play auxiliary roles and are dispensable for Hap1 activation by heme [40,41]. Heme promotes Hap1 transcriptional activity by enhancing the association of Hsp90 to the Hap1-Hsp70-Ydj1-Sro9 multichaperone complex, causing conformational changes in the complex [14,29,31,34,36,37,40,41]. Subsequently, the complex transforms into a conformationally different complex that has a high affinity for DNA binding and is transcriptionally active [14,29,31,34,36,37,40,41].…”

Regulation of the HAP1 gene involves positive actions of histone deacetylases

“…The DNA fragment containing −321 to −360 overlaps with the −341 to −380 DNA fragment and exhibited strong complex formation (lanes 19 and 20), although the percentage of bound vs. free was higher for the −341 to −380 DNA fragment. All longer DNA fragments containing the −341 to −380 region formed Hap1-dependent complexes (see lanes [7][8][9][10][11][12][13][14][15][16]. The DNA fragment containing the −380 to −461 region also formed a Hap1-dependent complex (lanes 17 and 18), but it was much weaker, compared to that formed on the −341 to −380 DNA fragment.…”

“…The mechanism of heme-dependent transcriptional activation by Hap1 has been well studied [4,[9][10][11]. Previous studies have shown that heme regulation of Hap1 activity requires molecular chaperones Hsp90, Hsp70 and its cochaperones Ydj1 and Sro9 [12][13][14][15][16]. Hsp70 and its cochaperones Ydj1 and Sro9 confer Hap1 repression at low heme levels, whereas Hsp90 promotes heme activation of Hap1 [12][13][14][15]17].…”