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.

FIG. 1

Analysis of the purified Hap1-associated proteins. (A) Yeast cell extracts containing His₆-Hap1 were purified by Ni-NTA columns, and the eluted fractions were analyzed with SDS–10% polyacrylamide gels. The eluted peak fraction (Elu; lane 1) and unpurified whole-cell extracts (Ext; lane 2) are shown. The positions of the protein weight markers (lane 3) are denoted. The two major bands (A and B) are marked and were subjected to mass spectrometric fragment analysis. (B) DNA-Hap1 complexes formed by purified proteins from Ni-NTA columns. The eluate was incubated with radiolabeled DNA in the absence (lane 1) and presence (lane 2) of 2 ng of heme per μl and then analyzed on a 3.5% polyacrylamide gel. The positions of the HMC and dimeric complex (DC) are marked.

FIG. 2

(A) Western blot showing Hap1 protein levels in wild-type and mutant ssa cells. Cell extracts (50 μg) prepared from wild-type (wt; lane 1) and a2a3a4 (lane 2) cells expressing Hap1 from a 2-μm plasmid (35) were analyzed on an SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with an antibody against Hap1. (B) Effect of Ssa proteins on Hap1 DNA binding. Yeast cell extracts were prepared from wild-type (lanes 3 and 4) and a2a3a4 (lanes 1 and 2) cells. Electrophoretic mobility shift assays were carried out. Extracts (20 μg) containing Hap1 prepared from yeast cells were incubated with radiolabeled DNA in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of 2 ng of heme per μl. The reaction mixtures were analyzed on 3.5% polyacrylamide gels. The positions of the HMC and dimeric complex (DC) are marked. (C) Protein binding at TEF2 UAS and CDEI sites in wild-type and mutant ssa cells. Extracts (20 μg) prepared from wild-type (lanes 2, 3, 5, and 6) or mutant a2a3a4 (lanes 1 and 4) cells were incubated with radiolabeled synthetic DNA containing TEF2 UAS (43) (lanes 1 to 3) or the CDEI site (3, 32) (lanes 4 to 6). In lanes 3 and 6, 1 μg of cold synthetic TEF2 UAS (lane 3) or CDEI site (lane 6) was included in the DNA-binding reactions, respectively. The reaction mixtures were analyzed on a 4% polyacrylamide gel.

FIG. 3

(A) Effect of low levels of Ssa on Hap1 DNA binding. Yeast 5B6 cells (Δssa1Δssa2 Δssa4 pGAL1-SSA1) were grown in medium containing 2% galactose (lanes 5 and 6) for a high Ssa expression level, 1% galactose plus 1% glucose (1% Gal; lanes 3 and 4) for a low (5 to 10% of high) Ssa expression level, or 0.25% galactose plus 1.75% glucose (0.25% Gal; lanes 1 and 2) for a very low (<2% of high) Ssa1 expression level. Cell extracts were prepared from these cells, and DNA-binding reactions were carried out in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of 2 ng of heme per μl. (B) Hap1 DNA binding in wild-type cells was unaffected by various amounts of galactose and glucose. Yeast wild-type JN55 cells were grown in medium containing 2% galactose (lanes 5 and 6), 1% galactose plus 1% glucose (lanes 3 and 4), or 0.25% galactose plus 1.75% glucose (lanes 1 and 2). Cell extracts were prepared from these cells, and DNA-binding reactions were carried out in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of 2 ng of heme per μl. (C) Protein binding at TEF2 UAS and CDEI sites in cells expressing various levels of Ssa. Extracts (20 μg) prepared from 5B6 cells grown in medium containing 2% galactose (lanes 1 and 4), 1% galactose plus 1% glucose (lanes 2 and 5), or 0.25% galactose plus 1.75% glucose (lanes 3 and 6) were incubated with radiolabeled synthetic DNA containing the CDEI site (3, 32) (lanes 1 to 3) or TEF2 UAS (43) (lanes 4 to 6). The reaction mixtures were analyzed on a 4% polyacrylamide gel.

FIG. 4

(A to H) Effects of low levels of Ssa on the composition of the DNA-bound Hap1 complexes. Yeast 5B6 cells (Δssa1 Δssa2 Δssa4 pGAL1-SSA1) were grown in medium containing 2% galactose (lane 3) (A to H), 1% galactose plus 1% glucose (lane 2) (A to H), or 0.25% galactose plus 1.75% glucose (lane 1) (A to H). Extracts were incubated with streptavidin-conjugated magnetic beads (Dynal) prebound with the biotinylated synthetic wild-type or mutant Hap1-binding site (18) in the absence of heme. The beads were extensively washed and boiled in SDS gel loading buffer to release the bound proteins (41). Bound proteins (B, D, F, and H) and proteins in the original extracts (A, C, E, and G) were subsequently electrophoresed on SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against Hap1 (A and B), Ssa (C and D), Hsp90 (E and F), and Ydj1 (G and H). No bound protein was detected when a mutated Hap1-binding site was used in the pull-down experiments. These experiments were repeated twice. (I) Western blot showing Hap1 protein levels in Δydj1 cells. Yeast MHY200Δydj1 cells expressing Hap1 and bearing the expression plasmid for wild-type Ydj1 (lane 1), the empty vector (lane 2), or the mutant F47L (lane 3) were prepared and subjected to Western blotting analysis with an anti-Hap1 antibody. The same result was obtained when Hap1-18 was expressed in the cells.