Vol. 12, No. 6
MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2616-2623
0270-7306/92/062616-08$02.00/0 Copyright © 1992, American Society for Microbiology
HAP1 and ROX1 Form a Regulatory Pathway in the Repression of HEM13 Transcription in Saccharomyces cerevisiae
Received 14 October 1991/Accepted 19 March 1992
HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression ofHEM13 in the absence of heme. It is also shown that the repression ofHEM13 transcription caused by heme involves the H4P] and ROXI gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROXI was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAPI-mediated regulation of ROXI transcription in response to heme levels followed by ROX1mediated repression of HEM13 transcription. In support of this model, expression of ROXI under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap] mutant strain. The ability of ROX1 encoded by the galactose-inducible ROXI construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROXI transcription. In the yeast Saccharomyces cerevisiae, expression of a number of genes is regulated in response to oxygen levels. Transcription of several genes is induced in the presence of oxygen (9, 15, 17, 19, 33, 36, 37, 39). Most of these genes encode cytochromes and respiratory enzymes. Other oxygen-induced genes include HMG1 and PUTI, which encode enzymes involved in sterol biosynthesis and utilization of proline as a nitrogen source; both of these metabolic pathways are oxygen dependent (36, 41). The effect of oxygen on the expression of many of these genes is mediated by heme. In yeast cells, heme is a molecule that is synthesized only in the presence of oxygen (23). Therefore, intracellular heme levels can serve as an internal indicator of oxygen levels in the environment. In addition to genes which are induced by oxygen, expression of a number of genes in yeast is repressed by oxygen or heme. These hypoxic genes include COXSb, ANBI (TIFSJB), and HMG2 (15, 21, 36, 39). COXSb encodes an isozyme of subunit V of cytochrome oxidase (4), ANBI encodes a protein which is homologous to translational initiation factor eIF-5A (24, 33a), and HMG2 encodes the sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (2). These three genes are alike in that each has a homolog which is regulated in the opposite manner by heme or oxygen. Expression of COXSa (15), TIFSJA (20, 33a), and HMGI (36) are induced by oxygen and heme. Four genes, HAPI to -4, have been found to encode gene products which are required for activation of gene expression in response to heme (5, 8, 12, 28). A4PI encodes a transcriptional activator, the DNA-binding activity of which is heme dependent (27). HAP2, HAP3, and H4P4 encode proteins which function as a heterotrimeric transcriptional complex to activate transcription from CCAAT sequences in the upstream regulatory regions of some heme-induced genes (5). The ROXI and REOI gene products are needed for repression of hypoxic or heme-repressed genes under aerobic conditions (15, 22, 38). Transcription of ROXI was found to be heme and oxygen dependent (22). Thus, the
ROXJ gene product is present only in aerobically grown cells and therefore can serve to repress expression of hypoxic genes only in cells grown under aerobic conditions. As heme biosynthesis is a process which contains two oxygen-dependent steps, it is of interest to determine whether expression of the heme biosynthetic enzymes is regulated by heme or oxygen levels and whether expression of these genes is under the control of the same regulatory factors which have been defined as necessary for transcription of the heme-regulated genes. It has already been shown that although HEM1 expression is not heme dependent, it requires the function of the HAP2/3/4 transcriptional complex (18). In this report, this subject of investigation is extended to consider regulation of HEM13 expression. HEM13 in S. cerevisiae encodes the enzyme coproporphyrinogen oxidase, which catalyzes the sixth step in the heme biosynthetic pathway (44). This enzyme utilizes oxygen as a substrate in the conversion of coproporphyrinogen III to protoporphyrinogen IX. Transcription of HEM13 is regulated by oxygen and heme (43, 44). Growth of cells under anaerobic conditions or in the absence of heme results in an increase in HEM13 mRNA levels. The effect of oxygen on expression of HEM13 is mediated via intracellular heme levels, as has been observed for ANBI and COX5b (15, 21). Thus, expression of HEM13 is regulated in the same manner as is expression of ANBI and COXSb. In this study, I attempt to define the components of the regulatory pathway that controls HEM13 expression and show that HAP) and ROXI gene products are essential components of the pathway that represses HEM13 expression in the presence of heme. The ROXI gene product is required for heme-dependent repression of HEMJ3 expression, and the HAP) gene product is needed for the activation of ROXI expression by heme.
MATERUILS AND METHODS Strains and media. Strains used in this study are listed in Table 1. Strain SC252a is a gift from R. Storms. Plasmid 2616
Downloaded from http://mcb.asm.org/ on May 9, 2015 by FU Berlin, Univ.-Klinikum Benjamin Franklin
TERESA KENG Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada
VOL. 12, 1992
REPRESSION OF HEM13 TRANSCRIPTION IN YEAST CELLS
2617
TABLE 1. Yeast strains Genotype
Source
SC252a TKY22 TKY24 1-7aheml::LEU2 LGWlheml::LEU2
MATa leu2-3 leu2-112 ura3-52 adel MATa leu2-3 leu2-112 ura3-52 adel trpl::hisG Aheml MATa leu2-3 leu2-112 ura3-52 adel trpl::hisG hapl ::LEU2 Aheml MATa leu2-3 leu2-112 his4-519 adel ura3-52 heml::LEU2 MATa leu2-3 leu2-112 his4-519 adel ura3-52 heml ::LEU2 hap2-1 M4Ta leu2-3 leu2-112 his4-519 adel ura3-52 heml::LEU2 hapl-l M4Ta trpl-J cycl-l his4-519 leu2-3 leu2-112 gal MATa trpl-1 cycl-1 his4-519 leu2-3 leu2-112 gal roxl::LEU2 MATa leu2-3 leu2-112 ura3-52 adel Aheml MATa trpl-l ura3-52 leu2-3 leu2-112 Aheml MATa his4-519 ura3-52 leu2-3 leu2-112 Aheml roxl::LEU2
R. Storms This study This study T. Prezant L. Guarente K. Pfeifer R. Zitomer R. Zitomer This study This study This study
TKY1l AH12-7 AH12-7Aroxl TKY18 TKY1-4c TKY1-5d
ANot-TthlllI was provided by M. Haldi and contains a deleted, nonfunctional heml gene on an integrative plasmid containing the URA3 marker (13). It was introduced into SC252a by integrative transformation after linearization with SalI. Ura- colonies were recovered by selection on 5-fluoroorotic acid plates and screened for heme prototrophy (3). The Aheml Ura- derivative was named TKY18. The hapl::LEU2 disruption vector was provided by K. Pfeifer (27). This disruption was introduced as a PstI fragment into the Aheml mutant strain, TKY18, by a one-step gene disruption method to yield strain TKY19 (31). The trpl disruption vector, pNKY1009, was a gift from E. Alani (1). The trpl disruption was introduced into strains TKY18 and TKY19 as a trpl::hisG-URA3-hisG mutation. Recombination between the hisG repeats resulted in strains containing a trpl::hisG disruption and recovery of the Ura- phenotype. The trpl ::hisG derivative of TKY18 was TKY22, and that of TKY19 was TKY24. Each of the disruptions was verified by Southern blot analysis of chromosomal DNA from the respective strains. AH12-7 and AH12-7Aroxl were generous gifts of R. Zitomer (22). Strains 1-7aheml::LEU2 and LGWlheml::LEU2 were provided by T. Prezant and L. Guarente (29), respectively. 1-7ahapl-lheml::LEU2 is renamed TKY11 and was obtained from K. Pfeifer. AH127Aroxl was crossed with TKY18, and the diploids formed were induced to sporulate. The resulting haploids were tested for their respective phenotypes. From this analysis, we obtained TKY1-4c, a Aheml haploid and TKY1-5d, a Ahemlroxl::LEU2 haploid. Yeast cells were grown in YEP or synthetic selective media as described by Sherman et al. (35). All amino acid and base supplements were added to a final concentration of 40 ,ug/ml. Glucose and galactose, when added, were used at 2%. Solid medium contained 2% agar. Strains which contained a heml mutation were defective in the enzyme b-aminolevulinate (8-ALA) synthase and could not make 8-ALA. Strains TKY22, TKY24, TKY1-4c, and TKY1-5d, which have such mutations, were grown in medium supplemented with 50 jig of b-ALA per ml or in the presence of 0.5% (vol/vol) Tween 80, 20 jig of ergosterol per ml, and 40 jig of methionine per ml (6). Tween 80-ergosterol was made up as a solution of 0.2% (wt/vol) ergosterol in Tween 80-ethanol (1:1). However, hemi mutant strain 1-7aheml:: LEU2 and related strains in the same genetic background will not grow in the absence of b-ALA. This strain was grown in the presence of high (50 ig/rml) or low (0.5 jig/ml) concentrations of b-ALA instead. hem] mutants grown in the presence of high levels of b-ALA were heme sufficient, while Aheml mutants grown in the presence of Tween 80,
ergosterol, and methionine or low levels of b-ALA were heme deficient (9). Plasmid constructions. Restriction digests and ligations were carried out under conditions recommended by the suppliers of the restriction and modification enzymes (Boehringer-Mannheim Canada, New England Biolabs, and Pharmacia Canada). Plasmids were propagated in Escherichia coli MC1061. Plasmid pTK13Z-T contains a HEM13-lacZ fusion on an ARS-CEN plasmid with a TRPI marker. Plasmid pBIOl (a gift of B. Osborne) is an integrating plasmid with a CYCllacZ fusion and a TRPI marker. The ARS1-CEN4 sequences were isolated as a HindIII 2.8-kb fragment from pBL101 (8) and inserted into the unique HindIII site in pBIOl to generate YCpBIO1. Plasmid pAM18XSX-B-K, a plasmid which contains the upstream regulatory sequences (UAS) of HEM13 and part of the HEM13 coding region in pAM18, was cut with XhoI, treated with Klenow enzyme to generate blunt ends, and digested with BamHI to release a 1.6-kb fragment (30). This fragment was used to replace the SmaIBamHI CYCl sequences in YCpBIOl to generate pTK13Z-T and the identical CYCI sequences in plasmid pBL101 to generate YCp13Z. These plasmids contain identical HEM13lacZ fusions with 1.5 kb of the upstream noncoding region and the first 34 codons of HEM13 fused to lacZ. Plasmid pTKAZ-T is an ARS-CEN plasmid with a TRPI marker which contains a CYCl-lacZ fusion fused to the regulatory sequences of the ANBI gene. It was constructed by replacing the SmaI-SalI fragment which represents the UAS of CYCI in YCpBIO1 with a 0.3-kb SmaI-XhoI fragment from plasmid pLG669Z (10). The resulting plasmid places the intact TATA and initiation sequences of CYCI under the control of the ANBI regulatory sequences. Expression of such a fusion construct has been demonstrated to be induced under anaerobic conditions (25). PPGALROX1 is a 2-jim plasmid with a URA3 marker which contains UASGAL fused to the ROXJ transcription unit. Plasmid YEpPGAL was first constructed by ligation of a 1.7-kb ClaI-BamiHI fragment from plasmid pLGSD5 which contains UASGAL and CYCl TATA and initiation sequences to the 6.27-kb ClaI-BamHI backbone of YEp24 (11). YEpPGAL was then digested with XhoI and PvuII, and the ends were rendered flush with Klenow fragment. The 5' phosphates were removed by treatment with calf intestine alkaline phosphatase. This 7.55-kb backbone contains the UASGAL and is deleted for the CYCJ sequences. The ROXI transcription unit was isolated as a 2.0-kb XbaI-HindIII fragment from YCpROX1 (22). (YCpROX1 was generously provided by R. Zitomer.) The ends of this fragment were
Downloaded from http://mcb.asm.org/ on May 9, 2015 by FU Berlin, Univ.-Klinikum Benjamin Franklin
Strain
2618
KENG
RESULTS Regulation of HEM13 expression by heme. It has been shown that HEM13 transcription is derepressed under anaerobic conditions (43, 44). This effect of oxygen on HEM13 expression is mediated via heme levels such that derepression of HEM13 expression can also be achieved in a heme biosynthetic mutant grown without heme as a supplement. The effects of heme on expression were investigated in heml mutant strains TKY22 and 1-7aheml::LEU2. heml mutants are defective in the first step of heme biosynthesis and fail to make f-ALA. Thus, one can control the intracellular heme levels of a heml mutant by controlling the amount of B-ALA added to the medium (9). heml mutants grown in the presence of high levels of 8-ALA are heme sufficient. Some heml mutants can also grow in the absence of f-ALA, in media supplemented with Tween 80 (supplying oleic acid), ergosterol, and methionine, products whose biosyntheses require heme or molecules which are derived from intermediates in the heme biosynthetic pathway (23). heml mutants grown in such media do not make heme and are heme deficient. Total RNA was isolated from two different heml mutant strains grown under heme-sufficient and heme-deficient conditions and analyzed in a Northern blot hybridization experiment. The results (Fig. 1) indicate that in both heml strains examined, HEM13 mRNA levels were low in cells grown in the presence of f-ALA and were induced in cells grown without or with low levels of f-ALA. I also examined the
TKY22 Ahem 1
1-7a
hem 1::LEU2 .tg/ml 6-ALA 0.5 50
8-ALA _
*
+
HEM13
*
ACTIN FIG. 1. Effect of heme on expression of HEM13. heml mutant strain TKY22 was grown in synthetic glucose medium in the presence or absence of f-ALA, and strain 1-7aheml::LEU2 was grown in the presence of 50 or 0.5 ,ug of f-ALA per ml. Total RNA was isolated and used in hybridization experiments; 20 ,ug of RNA was loaded per lane. The RNA filters were first hybridized with a radioactively labeled, random-primed probe prepared from a 2.2-kb DraI fragment from pHEM13-1. The blots were then stripped and reprobed with a labeled actin probe.
level of ,-galactosidase activity corresponding to expression of HEM13-lacZ fusion on a low-copy-number centromeric plasmid (pTK13Z-T or YCp13Z) in these two hem] mutant strains each grown under heme-sufficient or heme-deficient conditions. The HEM13-lacZ fusions present on these plasmids have been shown to contain the entire HEM13 regulatory region (30a). The results (Table 2) indicate that expression of HEM13 is low in cells grown in the presence of heme (1 to 2 Miller units) and that expression is derepressed 16- to 20-fold in strains 1-7aheml::LEU2 and TKY22. ROX1 represses HEM13 expression. The product of the ROX] gene is required for the repression of a number of anaerobic genes or genes that are normally expressed in the absence of oxygen (15, 21, 22). Strains carrying a Arox] allele express these hypoxia-inducible genes such as ANBI and COX5b under aerobic conditions (15, 21). Because HEM13 is an anaerobic gene, I wished to examine the effect of a Aroxl mutation on HEM13 expression. To this end, the levels of ,-galactosidase activity made from a HEM13-lacZ fusion in a Arox] strain and in an isogenic strain with a wild-type ROX1 allele were studied. The expression of an ANB]-CYCI-lacZ fusion in these two strains was also examined. The results (summarized in Table 3) indicate that expression of HEM13-lacZ is increased dramatically in a Atroxl strain. Expression is very low (
MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2616-2623
0270-7306/92/062616-08$02.00/0 Copyright © 1992, American Society for Microbiology
HAP1 and ROX1 Form a Regulatory Pathway in the Repression of HEM13 Transcription in Saccharomyces cerevisiae
Received 14 October 1991/Accepted 19 March 1992
HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression ofHEM13 in the absence of heme. It is also shown that the repression ofHEM13 transcription caused by heme involves the H4P] and ROXI gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROXI was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAPI-mediated regulation of ROXI transcription in response to heme levels followed by ROX1mediated repression of HEM13 transcription. In support of this model, expression of ROXI under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap] mutant strain. The ability of ROX1 encoded by the galactose-inducible ROXI construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROXI transcription. In the yeast Saccharomyces cerevisiae, expression of a number of genes is regulated in response to oxygen levels. Transcription of several genes is induced in the presence of oxygen (9, 15, 17, 19, 33, 36, 37, 39). Most of these genes encode cytochromes and respiratory enzymes. Other oxygen-induced genes include HMG1 and PUTI, which encode enzymes involved in sterol biosynthesis and utilization of proline as a nitrogen source; both of these metabolic pathways are oxygen dependent (36, 41). The effect of oxygen on the expression of many of these genes is mediated by heme. In yeast cells, heme is a molecule that is synthesized only in the presence of oxygen (23). Therefore, intracellular heme levels can serve as an internal indicator of oxygen levels in the environment. In addition to genes which are induced by oxygen, expression of a number of genes in yeast is repressed by oxygen or heme. These hypoxic genes include COXSb, ANBI (TIFSJB), and HMG2 (15, 21, 36, 39). COXSb encodes an isozyme of subunit V of cytochrome oxidase (4), ANBI encodes a protein which is homologous to translational initiation factor eIF-5A (24, 33a), and HMG2 encodes the sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (2). These three genes are alike in that each has a homolog which is regulated in the opposite manner by heme or oxygen. Expression of COXSa (15), TIFSJA (20, 33a), and HMGI (36) are induced by oxygen and heme. Four genes, HAPI to -4, have been found to encode gene products which are required for activation of gene expression in response to heme (5, 8, 12, 28). A4PI encodes a transcriptional activator, the DNA-binding activity of which is heme dependent (27). HAP2, HAP3, and H4P4 encode proteins which function as a heterotrimeric transcriptional complex to activate transcription from CCAAT sequences in the upstream regulatory regions of some heme-induced genes (5). The ROXI and REOI gene products are needed for repression of hypoxic or heme-repressed genes under aerobic conditions (15, 22, 38). Transcription of ROXI was found to be heme and oxygen dependent (22). Thus, the
ROXJ gene product is present only in aerobically grown cells and therefore can serve to repress expression of hypoxic genes only in cells grown under aerobic conditions. As heme biosynthesis is a process which contains two oxygen-dependent steps, it is of interest to determine whether expression of the heme biosynthetic enzymes is regulated by heme or oxygen levels and whether expression of these genes is under the control of the same regulatory factors which have been defined as necessary for transcription of the heme-regulated genes. It has already been shown that although HEM1 expression is not heme dependent, it requires the function of the HAP2/3/4 transcriptional complex (18). In this report, this subject of investigation is extended to consider regulation of HEM13 expression. HEM13 in S. cerevisiae encodes the enzyme coproporphyrinogen oxidase, which catalyzes the sixth step in the heme biosynthetic pathway (44). This enzyme utilizes oxygen as a substrate in the conversion of coproporphyrinogen III to protoporphyrinogen IX. Transcription of HEM13 is regulated by oxygen and heme (43, 44). Growth of cells under anaerobic conditions or in the absence of heme results in an increase in HEM13 mRNA levels. The effect of oxygen on expression of HEM13 is mediated via intracellular heme levels, as has been observed for ANBI and COX5b (15, 21). Thus, expression of HEM13 is regulated in the same manner as is expression of ANBI and COXSb. In this study, I attempt to define the components of the regulatory pathway that controls HEM13 expression and show that HAP) and ROXI gene products are essential components of the pathway that represses HEM13 expression in the presence of heme. The ROXI gene product is required for heme-dependent repression of HEMJ3 expression, and the HAP) gene product is needed for the activation of ROXI expression by heme.
MATERUILS AND METHODS Strains and media. Strains used in this study are listed in Table 1. Strain SC252a is a gift from R. Storms. Plasmid 2616
Downloaded from http://mcb.asm.org/ on May 9, 2015 by FU Berlin, Univ.-Klinikum Benjamin Franklin
TERESA KENG Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada
VOL. 12, 1992
REPRESSION OF HEM13 TRANSCRIPTION IN YEAST CELLS
2617
TABLE 1. Yeast strains Genotype
Source
SC252a TKY22 TKY24 1-7aheml::LEU2 LGWlheml::LEU2
MATa leu2-3 leu2-112 ura3-52 adel MATa leu2-3 leu2-112 ura3-52 adel trpl::hisG Aheml MATa leu2-3 leu2-112 ura3-52 adel trpl::hisG hapl ::LEU2 Aheml MATa leu2-3 leu2-112 his4-519 adel ura3-52 heml::LEU2 MATa leu2-3 leu2-112 his4-519 adel ura3-52 heml ::LEU2 hap2-1 M4Ta leu2-3 leu2-112 his4-519 adel ura3-52 heml::LEU2 hapl-l M4Ta trpl-J cycl-l his4-519 leu2-3 leu2-112 gal MATa trpl-1 cycl-1 his4-519 leu2-3 leu2-112 gal roxl::LEU2 MATa leu2-3 leu2-112 ura3-52 adel Aheml MATa trpl-l ura3-52 leu2-3 leu2-112 Aheml MATa his4-519 ura3-52 leu2-3 leu2-112 Aheml roxl::LEU2
R. Storms This study This study T. Prezant L. Guarente K. Pfeifer R. Zitomer R. Zitomer This study This study This study
TKY1l AH12-7 AH12-7Aroxl TKY18 TKY1-4c TKY1-5d
ANot-TthlllI was provided by M. Haldi and contains a deleted, nonfunctional heml gene on an integrative plasmid containing the URA3 marker (13). It was introduced into SC252a by integrative transformation after linearization with SalI. Ura- colonies were recovered by selection on 5-fluoroorotic acid plates and screened for heme prototrophy (3). The Aheml Ura- derivative was named TKY18. The hapl::LEU2 disruption vector was provided by K. Pfeifer (27). This disruption was introduced as a PstI fragment into the Aheml mutant strain, TKY18, by a one-step gene disruption method to yield strain TKY19 (31). The trpl disruption vector, pNKY1009, was a gift from E. Alani (1). The trpl disruption was introduced into strains TKY18 and TKY19 as a trpl::hisG-URA3-hisG mutation. Recombination between the hisG repeats resulted in strains containing a trpl::hisG disruption and recovery of the Ura- phenotype. The trpl ::hisG derivative of TKY18 was TKY22, and that of TKY19 was TKY24. Each of the disruptions was verified by Southern blot analysis of chromosomal DNA from the respective strains. AH12-7 and AH12-7Aroxl were generous gifts of R. Zitomer (22). Strains 1-7aheml::LEU2 and LGWlheml::LEU2 were provided by T. Prezant and L. Guarente (29), respectively. 1-7ahapl-lheml::LEU2 is renamed TKY11 and was obtained from K. Pfeifer. AH127Aroxl was crossed with TKY18, and the diploids formed were induced to sporulate. The resulting haploids were tested for their respective phenotypes. From this analysis, we obtained TKY1-4c, a Aheml haploid and TKY1-5d, a Ahemlroxl::LEU2 haploid. Yeast cells were grown in YEP or synthetic selective media as described by Sherman et al. (35). All amino acid and base supplements were added to a final concentration of 40 ,ug/ml. Glucose and galactose, when added, were used at 2%. Solid medium contained 2% agar. Strains which contained a heml mutation were defective in the enzyme b-aminolevulinate (8-ALA) synthase and could not make 8-ALA. Strains TKY22, TKY24, TKY1-4c, and TKY1-5d, which have such mutations, were grown in medium supplemented with 50 jig of b-ALA per ml or in the presence of 0.5% (vol/vol) Tween 80, 20 jig of ergosterol per ml, and 40 jig of methionine per ml (6). Tween 80-ergosterol was made up as a solution of 0.2% (wt/vol) ergosterol in Tween 80-ethanol (1:1). However, hemi mutant strain 1-7aheml:: LEU2 and related strains in the same genetic background will not grow in the absence of b-ALA. This strain was grown in the presence of high (50 ig/rml) or low (0.5 jig/ml) concentrations of b-ALA instead. hem] mutants grown in the presence of high levels of b-ALA were heme sufficient, while Aheml mutants grown in the presence of Tween 80,
ergosterol, and methionine or low levels of b-ALA were heme deficient (9). Plasmid constructions. Restriction digests and ligations were carried out under conditions recommended by the suppliers of the restriction and modification enzymes (Boehringer-Mannheim Canada, New England Biolabs, and Pharmacia Canada). Plasmids were propagated in Escherichia coli MC1061. Plasmid pTK13Z-T contains a HEM13-lacZ fusion on an ARS-CEN plasmid with a TRPI marker. Plasmid pBIOl (a gift of B. Osborne) is an integrating plasmid with a CYCllacZ fusion and a TRPI marker. The ARS1-CEN4 sequences were isolated as a HindIII 2.8-kb fragment from pBL101 (8) and inserted into the unique HindIII site in pBIOl to generate YCpBIO1. Plasmid pAM18XSX-B-K, a plasmid which contains the upstream regulatory sequences (UAS) of HEM13 and part of the HEM13 coding region in pAM18, was cut with XhoI, treated with Klenow enzyme to generate blunt ends, and digested with BamHI to release a 1.6-kb fragment (30). This fragment was used to replace the SmaIBamHI CYCl sequences in YCpBIOl to generate pTK13Z-T and the identical CYCI sequences in plasmid pBL101 to generate YCp13Z. These plasmids contain identical HEM13lacZ fusions with 1.5 kb of the upstream noncoding region and the first 34 codons of HEM13 fused to lacZ. Plasmid pTKAZ-T is an ARS-CEN plasmid with a TRPI marker which contains a CYCl-lacZ fusion fused to the regulatory sequences of the ANBI gene. It was constructed by replacing the SmaI-SalI fragment which represents the UAS of CYCI in YCpBIO1 with a 0.3-kb SmaI-XhoI fragment from plasmid pLG669Z (10). The resulting plasmid places the intact TATA and initiation sequences of CYCI under the control of the ANBI regulatory sequences. Expression of such a fusion construct has been demonstrated to be induced under anaerobic conditions (25). PPGALROX1 is a 2-jim plasmid with a URA3 marker which contains UASGAL fused to the ROXJ transcription unit. Plasmid YEpPGAL was first constructed by ligation of a 1.7-kb ClaI-BamiHI fragment from plasmid pLGSD5 which contains UASGAL and CYCl TATA and initiation sequences to the 6.27-kb ClaI-BamHI backbone of YEp24 (11). YEpPGAL was then digested with XhoI and PvuII, and the ends were rendered flush with Klenow fragment. The 5' phosphates were removed by treatment with calf intestine alkaline phosphatase. This 7.55-kb backbone contains the UASGAL and is deleted for the CYCJ sequences. The ROXI transcription unit was isolated as a 2.0-kb XbaI-HindIII fragment from YCpROX1 (22). (YCpROX1 was generously provided by R. Zitomer.) The ends of this fragment were
Downloaded from http://mcb.asm.org/ on May 9, 2015 by FU Berlin, Univ.-Klinikum Benjamin Franklin
Strain
2618
KENG
RESULTS Regulation of HEM13 expression by heme. It has been shown that HEM13 transcription is derepressed under anaerobic conditions (43, 44). This effect of oxygen on HEM13 expression is mediated via heme levels such that derepression of HEM13 expression can also be achieved in a heme biosynthetic mutant grown without heme as a supplement. The effects of heme on expression were investigated in heml mutant strains TKY22 and 1-7aheml::LEU2. heml mutants are defective in the first step of heme biosynthesis and fail to make f-ALA. Thus, one can control the intracellular heme levels of a heml mutant by controlling the amount of B-ALA added to the medium (9). heml mutants grown in the presence of high levels of 8-ALA are heme sufficient. Some heml mutants can also grow in the absence of f-ALA, in media supplemented with Tween 80 (supplying oleic acid), ergosterol, and methionine, products whose biosyntheses require heme or molecules which are derived from intermediates in the heme biosynthetic pathway (23). heml mutants grown in such media do not make heme and are heme deficient. Total RNA was isolated from two different heml mutant strains grown under heme-sufficient and heme-deficient conditions and analyzed in a Northern blot hybridization experiment. The results (Fig. 1) indicate that in both heml strains examined, HEM13 mRNA levels were low in cells grown in the presence of f-ALA and were induced in cells grown without or with low levels of f-ALA. I also examined the
TKY22 Ahem 1
1-7a
hem 1::LEU2 .tg/ml 6-ALA 0.5 50
8-ALA _
*
+
HEM13
*
ACTIN FIG. 1. Effect of heme on expression of HEM13. heml mutant strain TKY22 was grown in synthetic glucose medium in the presence or absence of f-ALA, and strain 1-7aheml::LEU2 was grown in the presence of 50 or 0.5 ,ug of f-ALA per ml. Total RNA was isolated and used in hybridization experiments; 20 ,ug of RNA was loaded per lane. The RNA filters were first hybridized with a radioactively labeled, random-primed probe prepared from a 2.2-kb DraI fragment from pHEM13-1. The blots were then stripped and reprobed with a labeled actin probe.
level of ,-galactosidase activity corresponding to expression of HEM13-lacZ fusion on a low-copy-number centromeric plasmid (pTK13Z-T or YCp13Z) in these two hem] mutant strains each grown under heme-sufficient or heme-deficient conditions. The HEM13-lacZ fusions present on these plasmids have been shown to contain the entire HEM13 regulatory region (30a). The results (Table 2) indicate that expression of HEM13 is low in cells grown in the presence of heme (1 to 2 Miller units) and that expression is derepressed 16- to 20-fold in strains 1-7aheml::LEU2 and TKY22. ROX1 represses HEM13 expression. The product of the ROX] gene is required for the repression of a number of anaerobic genes or genes that are normally expressed in the absence of oxygen (15, 21, 22). Strains carrying a Arox] allele express these hypoxia-inducible genes such as ANBI and COX5b under aerobic conditions (15, 21). Because HEM13 is an anaerobic gene, I wished to examine the effect of a Aroxl mutation on HEM13 expression. To this end, the levels of ,-galactosidase activity made from a HEM13-lacZ fusion in a Arox] strain and in an isogenic strain with a wild-type ROX1 allele were studied. The expression of an ANB]-CYCI-lacZ fusion in these two strains was also examined. The results (summarized in Table 3) indicate that expression of HEM13-lacZ is increased dramatically in a Atroxl strain. Expression is very low (
