The Yeast Heme-responsive Transcriptional Activator Hap1 Is a Preexisting Dimer in the Absence of Heme

Abstract

In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer. In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer. Dimerization is a common mechanism by which the activity of numerous important biological macromolecules can be regulated. These molecules include receptors for growth hormones (1Kelly P.A. Postel-Vinay M.C. Finidori J. Edery M. Sotiropoulos A. Goujon L. Esposito N. Acta Paediatr. 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Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar) are regulated by Hsp90 and Hsp70 molecular chaperones. In particular, Hap1 is a heme-responsive transcriptional activator, which promotes transcription of genes required for respiration and for controlling oxidative damage in response to oxygen/heme (18Creusot F. Verdiere J. Gaisne M. Slonimski P.P. J. Mol. Biol. 1988; 204: 263-276Crossref PubMed Scopus (104) Google Scholar, 19Pfeifer K. Kim K.S. Kogan S. Guarente L. Cell. 1989; 56: 291-301Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 20Zitomer R.S. Lowry C.V. Microbiol. Rev. 1992; 56: 1-11Crossref PubMed Google Scholar). In the absence of heme, Hap1 is bound to cellular proteins including Hsp82 (the yeast homologue of Hsp90) and Ydj1, forming a higher order complex termed HMC 1The abbreviations used are: HMC, higher molecular weight complex; PAGE, polyacrylamide gel electrophoresis; DC, dimeric Hap1 complex; GST, glutathione S-transferase; NTA, nitrilotriacetic Acid (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar, 21Fytlovich S. Gervais M. Agrimonti C. Guiard B. EMBO J. 1993; 12: 1209-1218Crossref PubMed Scopus (55) Google Scholar, 22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). Hap1 DNA binding and transcriptional activities are repressed in this complex. Heme disrupts the HMC and permits Hap1 to bind to DNA as a dimer with high affinity, thereby activating transcription. The formation and disruption of the HMC are the key events in Hap1 repression in the absence of heme and subsequent activation by heme. How does the HMC repress Hap1, and how does the disassembly of the HMC lead to Hap1 activation? In the case of steroid hormone receptors, a plethora of reports suggested a model for how molecular chaperones control their activities (23Pratt W. Toft D. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar, 24Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (416) Google Scholar, 25Bohen S.P. Kralli A. Yamamoto K.R. Science. 1995; 268: 1303-1304Crossref PubMed Scopus (183) Google Scholar). In the absence of hormone, steroid hormone receptors form heteromeric complexes containing a receptor monomer, a dimer of Hsp90, and several other proteins including Hsp70 and Ydj1 (23Pratt W. Toft D. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar, 24Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (416) Google Scholar, 25Bohen S.P. Kralli A. Yamamoto K.R. Science. 1995; 268: 1303-1304Crossref PubMed Scopus (183) Google Scholar, 26Johnson J. Corbisier R. Stensgard B. Toft D. J. Steroid Biochem. Mol. Biol. 1996; 56: 31-37Crossref PubMed Scopus (66) Google Scholar). Hormone binding causes the dissociation of Hsp90 and other proteins, permitting steroid hormone receptors to dimerize and bind to DNA with high affinity and activate transcription (23Pratt W. Toft D. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar, 24Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (416) Google Scholar, 25Bohen S.P. Kralli A. Yamamoto K.R. Science. 1995; 268: 1303-1304Crossref PubMed Scopus (183) Google Scholar). This suggests that dimerization is an important event leading to the activation of steroid hormone receptors. In the case of Hap1, we envisioned two ways by which Hap1 activity could be controlled by the HMC. First, Hap1, like steroid hormone receptors, could exist as a monomer in the HMC and is thus repressed. When the HMC disassembles in the presence of heme, Hap1 is free to dimerize and bind to DNA with high affinity, thereby activating transcription. Second, Hap1 could already be a dimer in the HMC, but its activity is inhibited by molecular chaperones in the HMC. The disruption of the HMC by heme would then relieve this inhibition, thereby leading to Hap1 activation. Previous experiments indicated that the Hap1 dimerization domain plays a role in heme regulation (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar); however, it is not clear whether it acts by controlling dimerization or by making molecular interactions critical for Hap1 activity. In this report we describe a series of experiments aimed at distinguishing whether Hap1 is monomeric or oligomeric in the HMC. First, the two size variants of Hap1 coeluted on affinity columns in the absence of heme. Second, DNA mobility shift assays showed that the HMC recognizes various Hap1-binding sites in a manner similar to Hap1 dimer binding but completely different from monomer binding. Third, increasing overexpression of Hap1 caused graded increases in Hap1 DNA binding and transcriptional activation in the absence of heme, indicating that overexpression of Hap1 can functionally titrate out molecular chaperones. Together, these results strongly suggest that in the absence of heme, Hap1 is a preexisting oligomer in the HMC. Cells used for β-galactosidase assays and for extract preparation were grown as described previously (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar). Yeast strains used were MHY200 (MATaura3–52 leu2–3, 112 his4–519 ade1–100 hem1-Δ100 hap1::LEU2) (27Haldi M.L. Guarente L. Mol. Gen. Genet. 1995; 248: 229-235Crossref PubMed Scopus (20) Google Scholar), L51 (MAT a leu2–2, 2–112 his4–519 ade1–100 ura3–52 hap1::LEU2 trp1::HISG), and JEL1 (MATα leu2 trp1 ura3–52 nprb1–1122 pep4–3 ΔHis3::pGAL10-Gal4). To construct the expression plasmids for His6-Hap1 and His6-Hap1ΔKpn, a DNA fragment encoding Met-Arg-Gly-Ser-His-His-His-His-His-His was synthesized and inserted into the SamI site of the Hap1 and Hap1ΔKpn expression plasmids, SD5-HAP1 and SD5-HAP1ΔKpn (28Turcotte B. Guarente L. Genes Dev. 1992; 6: 2001-2009Crossref PubMed Scopus (41) Google Scholar). The resulting plasmids express Hap1 and Hap1ΔKpn with the MRGS-His6 tag at their N termini. To construct GST-Hap1, a DNA fragment encoding GST was inserted into theSamI site of the Hap1 expression plasmid, SD5-HAP1. The correct clones were identified by restriction digestion and confirmed by sequencing. Yeast MHY200 cells, with theHEM1 (encoding for δ-aminolevulinate synthase, the first enzyme in heme synthesis) (27Haldi M.L. Guarente L. Mol. Gen. Genet. 1995; 248: 229-235Crossref PubMed Scopus (20) Google Scholar) and HAP1 genes deleted, were transformed with yeast high copy 2 μm replicating plasmid expressing wild type Hap1, GST-Hap1, or His6-Hap1 from theGAL1–10 promoter and a UAS1/CYC1-TATA-lacZ reporter plasmid as described previously (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar, 22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). Cells were grown in synthetic complete medium containing 2% raffinose with limiting amounts of heme precursor δ-aminolevulinate (2 μg/ml) or high amounts of 5-aminolevulinate (250 μg/ml) to approximately A 0.5. Cells were then induced with 2% galactose for 7 h or for specified times (see Fig. 5) and harvested for determination of β-galactosidase as described previously (22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). Yeast cell extracts were to previously (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar, 22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). yeast expression plasmids were grown to A and induced with 2% galactose for h or for specified times (see Fig. Cells were harvested and in cell of Cells were then by with cell of and extracts were as described K. Guarente L. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). of extracts were determined by DNA binding were out in a with of μm as described (22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). of and extracts or containing approximately of proteins were used in The were at for h and then or polyacrylamide in for gel at The of the HMC and dimeric complex was by the To the HMC or the of Hap1 and His6-Hap1ΔKpn, were in a and with containing extracts were the at the of approximately were with of the containing The HMC complexes were with containing The was on and by and DNA mobility shift cell extracts and the from columns were first on and then to or Hap1 was by a the Hap1 DNA binding and a as described previously (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar). To the HMC and to whether it a Hap1 monomer or we the complex. We the GST or tag to the Hap1 N and determined how Hap1 to heme and how form the HMC. in His6-Hap1 and to heme in the as showed a of activity in the absence of heme, and heme their activity. Hap1, and His6-Hap1 a high molecular weight complex and bound to DNA with affinity Heme this complex and Hap1 to bind to DNA as a dimer with high These results show that of and GST to Hap1 on heme of Hap1 proteins and HMC the Hap1 can be used to the HMC. We by and We extracts from expressing a high of and His6-Hap1 from theGAL1–10 We then the extracts glutathione or We that and His6-Hap1 were on the but bound could not be for the bound His6-Hap1 complexes could be by in Fig. Hap1 was in the with and several other proteins were coeluted with Hap1 (see 17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar). These proteins also with His6-Hap1 on a and were to His6-Hap1 17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google and data not but were not on the when extracts containing Hap1 were These results strongly suggest that these proteins were bound to Hap1, not to The two proteins, A. Hach and L. and are in a of to Further, as the HMC bound to DNA with heme the HMC and Hap1 to bind to DNA with high affinity least This the previous that Hap1 DNA binding activity is repressed in the HMC (17Zhang L. Hach A. Wang C. Mol. Cell. Biol. 1998; 18: 3819-3828Crossref PubMed Scopus (90) Google Scholar, 21Fytlovich S. Gervais M. Agrimonti C. Guiard B. EMBO J. 1993; 12: 1209-1218Crossref PubMed Scopus (55) Google Scholar, L. Guarente L. 1994; PubMed Google Scholar). the for the HMC, we whether Hap1 is a in the HMC by We that Hap1 exists as an oligomer in the HMC, two different Hap1 size variants be in the HMC and thus on the that form of the size Hap1 exists as a monomer in the HMC, two Hap1 size variants not be and not on the all Hap1 for the activation domain are required for forming a HMC, we used Hap1 but not in the activation and Hap1 as two size Hap1 size variants were not used of in and expressing Hap1 We and Hap1 in yeast and extracts from these the that all Hap1 forms the HMC (22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar). We then the extracts containing and Hap1 a a we extracts containing Hap1 a to be at a higher Fig. and We then these columns and with We that Hap1 coeluted with His6-Hap1ΔKpn, and the of Hap1 to in the was similar to the in extracts Further, Hap1 was in the when the extracts not contain that the of Hap1 on the was not to binding to These results suggest that Hap1 is in a dimeric or higher oligomeric form in the absence of heme in the HMC. The suggests that Hap1 is a preexisting dimer or a higher oligomer in the HMC in the absence of heme. We this by whether Hap1 subunits in the HMC are to as a dimer. We the manner by which the HMC to various DNA Hap1 a C6 zinc cluster that recognizes a CGG triplet L. Guarente L. Genes Dev. 1994; 8: PubMed Scopus Google Scholar). A Hap1 dimer cooperatively to DNA sites containing a of CGG L. Guarente L. Genes Dev. 1994; 8: PubMed Scopus Google Scholar, L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar). The two C6 zinc are in to the two CGG in a L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar, L. Guarente L. R. Nat. Struct. Biol. 6: PubMed Scopus Google Scholar). of the to Hap1 binding affinity L. Guarente L. Genes Dev. 1994; 8: PubMed Scopus Google Scholar). when Hap1 is or that it form a dimer, it to DNA as a monomer with affinity L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar). These previous studies that the affinity of Hap1 monomeric binding is whether or CGG are in the DNA sites L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar), a Hap1 monomer can with CGG The DNA binding affinity of the HMC from monomer binding the HMC a Hap1 monomer or from dimer binding to the by molecular chaperones. the HMC Hap1 it bind to various Hap1-binding and sites with or CGG the HMC two Hap1 subunits and the C6 zinc are to it bind to various DNA sites in a manner similar to the Hap1 dimer in the presence of heme L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar). we determined and the ways by which the HMC and Hap1 dimer bind to various Hap1-binding and These sites include and a site containing a of and a of and and a Hap1 7 and a site with the CGG of to and a site with the in the to and and a site that in and L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar). Fig. that the HMC not bind to these strongly the that the HMC a Hap1 the HMC and Hap1 dimer bound to these sites in a similar The HMC binding was at the and the the affinity of Hap1 dimer binding is to be high L. Guarente L. Genes Dev. 1994; 8: PubMed Scopus Google L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar) the dimer binding was and HMC binding was by HMC and Hap1 dimer was not at the and sites that contain CGG showed that the of the HMC to the as the of the Hap1 dimer at these The data strongly suggest that the HMC, like the Hap1 dimer in the presence of heme, a complete site containing a of two CGG for that the HMC recognizes DNA as a Hap1 dimer, not a To this we out We the of various DNA sites on HMC and Hap1 dimer binding to the site We used and to with the binding of HMC and Hap1 dimer to the The completely DNA binding by the HMC and Hap1 dimer and with and with the binding by the HMC and Hap1 dimer to 7 and and and and not with the binding at and and showed that these with the HMC and dimer binding to the These results showed that the HMC and the Hap1 dimer when binding to various DNA These DNA binding experiments (see and strongly suggested that the HMC, like the Hap1 dimer in the presence of heme, cooperatively to DNA as a dimer. The results suggest that the HMC is a preexisting Hap1 dimer, bound and repressed by molecular chaperones. we that heme the dissociation of molecular chaperones in the HMC, thereby leading to Hap1 activation. To this we to Hap1 in the presence of heme. this was not heme strongly to the columns is to be a and it to all of we including When extracts were the in the presence of heme, of the heme to the on the of the and in the HMC. We also to Hap1 from the HMC by Hap1 from yeast Hap1 is and we were not to Hap1 from the columns (the the these we that overexpression of Hap1 functionally out the molecular chaperones in the HMC the Hap1 Hap1 DNA binding activity First, and HMC Second, the of the HMC its the molecular chaperones were and Hap1 the molecular dimeric Hap1 complex Further, as the Hap1 expression Hap1 transcriptional activity in in the absence of heme Hap1 activity a higher the transcription the that the (see Fig. and Fig. this of activity was Hap1 activity in the presence of heme (see Fig. Together, these results suggest that of molecular chaperones in the HMC is functionally to Hap1 transcriptional activation. In this we to the mechanism by which Hap1 is repressed in the HMC. We that Hap1 is a preexisting dimer or higher oligomer in the HMC in the absence of heme. First, Hap1 and coeluted on the in the absence of heme Second, DNA binding of the HMC showed that the HMC to various DNA sites in a manner similar to Hap1 dimer binding but not monomer binding L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar) (see and Third, increasing overexpression of Hap1 to DNA the formation of the Hap1 dimeric and a of Hap1 transcriptional activity in the absence of heme. These results suggest that Hap1 repression in the absence of heme is at least in part, to the of DNA binding of the preexisting Hap1 dimer by molecular chaperones. the HMC was not a preexisting dimer in the absence of heme, and heme were required for Hap1 then overexpression of Hap1 would not lead to the formation of the dimeric complex. Further, the the of Hap1 transcriptional activity in and the of Hap1 DNA binding activity from the HMC and in suggests that the of molecular chaperones is functionally important for Hap1 activation. it was that in the absence of heme, Hap1 is from dimerization by other proteins in the it bind to DNA with high affinity and activate transcription (22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google L. B. Guarente L. Proc. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). Heme disrupts the HMC, Hap1 is free to dimerize and thus bind to DNA with high affinity, thereby activating transcription (22Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar, L. B. Guarente L. Proc. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). Our data this dimerization The data show that in the absence of heme, Hap1 as a dimer or higher Further, the two subunits of the preexisting dimer act cooperatively in DNA the of with CGG triplet completely HMC binding. of the of molecular the DNA binding affinity of the HMC is with that of the Hap1 dimer in the presence of heme (see The DNA binding affinity of the HMC from dimer or In the two Hap1 subunits of the HMC are in with other be to the two CGG by L. Guarente L. Genes Dev. 1994; 8: PubMed Scopus Google Scholar, L. Guarente L. EMBO J. 1996; PubMed Scopus Google Scholar). these results suggest a model for how heme Hap1 activity. In the absence of heme, Hap1 exists as a dimer in the HMC, but its DNA binding and activities are inhibited by molecular chaperones. When heme to Hap1, it certain Hap1 the interactions on the preexisting Hap1 dimer by molecular chaperones in the HMC. the inhibition on Hap1 is and the Hap1 dimer to bind to DNA with high affinity, thereby leading to Hap1 activation. This model that dissociation of molecular chaperones in the HMC lead to Hap1 activation. data in Fig. suggest that molecular chaperones can be functionally out by overexpression of Hap1, and this to increases in Hap1 DNA binding and transcriptional activities. This model a of how the activity of a dimeric transcription can be the activity of other dimeric transcription such as and Pdr1 of the Gal4 is also regulated in this