The Hsp70-Ydj1 Molecular Chaperone Represses the Activity of the Heme Activator Protein Hap1 in the Absence of Heme

Abstract

In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

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].…”

“…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]. Hap1 transcriptional activation is conferred via the acidic activation domain near the C-terminus of Hap1 [35].…”

Regulation of the HAP1 gene involves positive actions of histone deacetylases

“…Radioactivity of the interested bands was visualized and quantified by using the PhosphoImage ™ system (Molecular Dynamics). DNA pull-down was performed as described previously [13,15]. Pulled-down proteins were analyzed by SDS-PAGE, followed by Western blotting analysis.…”

“…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.…”

“…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].…”