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Molecular and Cellular Biology, June 1999, p. 4324-4333, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A New Class of Repression Modules Is Critical for
Heme Regulation of the Yeast Transcriptional Activator Hap1
Angela
Hach,
Thomas
Hon, and
Li
Zhang*
Department of Biochemistry, NYU Medical
Center, New York, New York 10016
Received 18 December 1998/Returned for modification 8 February
1999/Accepted 24 March 1999
 |
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 244is 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.
| |
INTRODUCTION |
Heme plays a central role in oxygen
sensing and utilization in all living organisms. It not only serves as
a prosthetic group in numerous enzymes and proteins that use,
transport, and store oxygen, but it also directly regulates various
molecular and cellular processes for systems that use or sense oxygen
(18). For example, heme stimulates erythroid cell
differentiation, hepatic cell differentiation, and nerve cell
differentiation (24, 25). Heme promotes the transcription of
genes encoding globin chains in erythroid cells and genes encoding
cytochromes in hepatic cells (2, 5, 23), controls protein
synthesis through the heme-regulated inhibitor (HRI) kinase in
reticulocytes (3, 4), and regulates the assembly and
degradation of many protein or enzyme complexes, such as hemoglobin,
heme lyase, and 
-aminolevulinate (ALA) synthase (ALAS)
(13, 14, 18, 26). How does heme regulate these diverse
processes? Recent evidence suggests that heme regulates different
processes through a short sequence motif, the heme-responsive motif
(HRM) (14, 33). HRMs are found in heme-regulated proteins of
diverse functions, such as the Saccharomyces cerevisiae
heme-responsive transcriptional activator Hap1 (33), NF-E2
(16), HRI kinase (3, 4), the leader sequence of
ALAS (14), heme lyase (26) and heme oxygenase
(17).
Hap1 is a transcriptional activator whose activity is directly and
stringently controlled by heme. It activates transcription of genes
encoding functions required for respiration and for controlling oxidative damage in the yeast S. cerevisiae (36).
As the heme concentration rises, Hap1 activity increases accordingly
and reaches its limit at micromolar heme concentrations
(31). Hap1 contains five known functional domains (20,
30, 33, 34): the Zn2-Cys6 cluster, the dimerization
domain, the heme domain, the HRM7 domain, and the activation domain.
Recent experiments show that three of these domainsthe dimerization
domain, the heme domain, and the HRM7 domainare all important for
heme regulation (35). Two of these heme regulatory domains
contain HRMs: six are located in the heme domain, and one is in the
HRM7 domain (33). Deletion of the heme domain renders Hap1
constitutively active and unresponsive to heme (6, 32, 35).
Moreover, a synthetic peptide containing one HRM was shown to bind
directly to heme in vitro (33). These results suggest that
HRMs in Hap1 and other hemoproteins could bind to heme and mediate heme
regulation. However, recent experiments from various laboratories have
generated conflicting results on the functional role of HRMs. For
example, while the HRMs in heme lyase are critical for heme binding and
enzymatic activity (26), the HRMs in heme oxygenase and
NF-E2 are not important for protein functions (16, 17). In
Hap1, the role of HRMs is inferred from the existence of HRM1 to HRM6
in the heme domain, but there is no direct functional evidence to
demonstrate the role of HRMs in vivo. Thus, the role of HRMs in heme
regulation must be rigorously ascertained. In addition, the three heme
regulatory domains span a large internal Hap1 region from amino acid
position 117 to 1308, and one of these domains, the dimerization
domain, does not contain any HRM (35). Therefore, very
likely, unidentified modules in these domains also play important roles
in heme regulation. Furthermore, previous evidence suggests that heme
regulation of Hap1 involves the disassembly of a higher-order complex,
termed the high-molecular-weight complex (HMC) (6, 32, 35).
When the cellular heme concentration falls, Hap1 is bound by proteins,
including Hsp90 and Ydj1, and forms an HMC (35). Heme
disrupts the HMC and allows Hap1 to bind to DNA with high affinity
(6, 32, 35). Two lines of evidence strongly support the idea
that HMC formation is linked to Hap1 repression in the absence of heme.
First, the HMC binds to DNA with very low affinity. As the heme
concentration increases in vitro, the HMC is gradually transformed into
a smaller, dimeric Hap1 complex with high DNA-binding affinity (6,
32). This transformation correlates with the gradual increase of
Hap1 activity in response to higher heme concentrations in vivo
(31, 32, 35). Second, previously characterized Hap1 mutants
defective in HMC formation are also derepressed and gain a high level
of activity in the absence of heme (35). Until now, the Hap1
elements that are critical for HMC formation and Hap1 repression have
been largely unknown.
In this report, we describe a systematic functional analysis aimed at
defining Hap1 elements critical for heme regulation. We have also begun
to dissect the complex molecular interactions responsible for Hap1
repression in the absence of heme and for subsequent Hap1 activation by
heme. We found that two classes of elements are important for heme
regulation. One class is composed of three newly identified repression
modules that mediate Hap1 repression in the absence of heme. Another
class is composed of the previously identified HRMs, in particular
HRM7, which mediate heme activation. Analysis of the HMCs formed by
Hap1 mutants suggests that the repression modules mediate Hap1
repression, at least in part, by the formation of the HMC. These
results provide significant new insights into mechanisms of heme
regulation and transcriptional activation.
| |
MATERIALS AND METHODS |
Yeast strains and reporters.
The yeast strains used in this
study were MHY200 (MATa ura3-52 leu2-3,112 his4-519
ade1-100 hem1-
100 hap1::LEU2) (10), L51
(MATa ura3-52 leu2-3,112 his4-519 ade1-100
hap1::LEU2 trp1::HisG) (31), and
JEL1 (MAT
leu2 trp1 ura3-52 nprbl-1122 pep4-3
His3::pGAL10-GAL4). The UAS1/CYC1-lacZ
reporter plasmid has been described previously (27).
Construction of deletion mutants in the Hap1 heme domain.
To
construct Hap1 heme domain deletion mutants, DNA fragments containing
the coding sequences for amino acid residues in the corresponding
regions of both the DNA-binding domain and the heme domain in HH4-HH
(Fig. 1) were generated by PCR amplification, cleaved with the
BamHI restriction enzyme, and inserted into the BamHI site of the Hap1 expression plasmid
SD5-HAP1 (27). The expression plasmid for heme
was generated as previously described (35). Correct clones
were identified by restriction digestion and confirmed by sequencing.
Generation of deletion mutants in the HRM7 domain and the
dimerization domain.
To construct the expression plasmids for the
Hap1 mutants with residues 447 to 1309 or 746 to 1309 deleted (H7-d1
and H7-d2, Fig. 2), the plasmid pHAP1(447-1309) or
pHAP1(746-1309) (20) was cleaved with
BstEII and KpnI. The fragment containing
HAP1 sequences was then inserted into the
BstEII-KpnI site of SD5-HAP1.
To construct the other deletion mutants and H7-1 shown in Fig. 2, the
BglII-KpnI fragment encoding Hap1 residues 746 to
1309 from the HAP1 expression vector (SD5-HAP1)
(27) was inserted into a vector derived from the Stratagene
Bluescript II KS(+) vector (a BglII site was inserted into
the NotI site). Single-stranded uracil DNA was then
generated in the dut ung strain CJ 236 (Bio-Rad). Oligonucleotides encoding mutated amino acid residues, with 16 bases
complementary to the region on either side of the deleted or mutated
sequence, were annealed to the single-stranded uracil DNA.
Double-stranded DNA was synthesized and transformed into the wild-type
MV 1190 strain, and plasmid DNA was isolated from the transformants.
Deletion mutants were identified by restriction digestion. A
HaeII site was created at the mutated region in H7-1 and
used to identify the mutant. BglII-KpnI fragments
from confirmed mutants were inserted into SD5-HAP1 to
reconstruct the expression plasmids for the mutants. Mutant H7-2 was
generated by inserting the BglII-KpnI fragment
released from H7-1 into the HH0 plasmid (see Fig. 1) cleaved with
BglII and KpnI. Mutants were further confirmed by
DNA sequencing. Mutants with deletions in the dimerization domain (see
Fig. 3) were generated in the same manner except that a KS(+) vector
containing the BamHI fragment of SD5-HAP1
(27) was used in the mutagenesis. Oligonucleotide sequences
are available upon request.
Preparation of yeast extracts and DNA mobility shift assays.
Extracts were prepared according to previously established protocols
(32, 35). Briefly, yeast L51 or JEL1 cells bearing expression plasmids were grown to an optical density of 0.5 and induced
with 2% galactose for specified times (see the figure legends). Cells
were harvested and resuspended in 3 packed cell volumes of buffer (20 mM Tris, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM
dithiothreitol, 0.3 M NaCl, 1 mM phenylmethylsulfonyl fluoride, pepstatin [1 µg/ml], leupeptin [1 µg/ml]). Cells were then
permeabilized by agitation with 4 packed cell volumes of glass beads,
and extracts were collected as described previously (21).
This method consistently generated extracts with protein concentrations
of approximately 10 mg/ml.
DNA-binding reactions were carried out in a 20-µl volume with 5%
glycerol, 4 mM Tris (pH 8), 40 mM NaCl, 4 mM MgCl
2, 10 mM
dithiothreitol 3 µg of salmon sperm DNA, 10 µM ZnOAc
2,
and 300
µg of bovine serum albumin per ml as described previously
(
32).
Approximately 0.01 pmol of labeled
UAS1/
CYC1 or UAS/
CYC7 and 20
µg of protein
extracts were used in each reaction. The reaction
mixtures were
incubated at room temperature for 1 h and then loaded
onto 3.5%
polyacrylamide gels in 1/3× Tris-borate-EDTA (1/3TBE)
for
electrophoresis at 4°C. The intensity of bands representing
the HMCs
and dimeric complexes was quantified by using the PhosphoImage
system
(Molecular
Dynamics).
Western blotting.
Whole-cell extracts for Western blotting
were first separated on sodium dodecyl sulfate (SDS)-7%
polyacrylamide gels and then transferred to polyvinylidene difluoride
or nitrocellulose membranes. Hap1 was visualized by using a purified
antibody against glutathione S-transferase-Hap1 (GST-Hap1)
(residues 1 to 171) and a chemiluminescence Western blotting kit
(Boehringer Mannheim), as described previously (35).
-Galactosidase assays.
To determine -galactosidase
levels from reporter genes in cells containing wild-type Hap1 and
mutants in the presence and absence of heme, yeast high-copy-number
2µm replicating plasmids expressing wild-type Hap1 and mutants from
the GAL1-10 promoter were transformed into the strain MHY200
(10) bearing the UAS1/CYC1-lacZ reporter. Cells
were grown in synthetic complete medium containing 2% raffinose with
limiting amounts of heme precursor ALA (2 µg/ml) or high amounts
of ALA (250 µg/ml) to an optical density of approximately 0.5. Cells were then induced partially with 0.25% galactose-1.75% glucose
or 1% galactose-1% glucose or completely with 2% galactose for
7 h and harvested for determination of -galactosidase activity as described previously (32).
| |
RESULTS |
Deletion analysis shows that residues 245 to 278, not the HRMs in
the heme domain, are responsible for mediating heme regulation.
Hap1 contains seven HRMs: six clustered in the heme domain and one near
the activation domain. Interestingly, three copies of a 17-amino-acid
repeat are found in the heme domain between HRM2 and -3, HRM3 and -4, and HRM4 and -5 (Fig. 1). We postulated that, in vivo, the HRMs are responsible for heme binding, whereas the
17-amino-acid repeats provide interaction surfaces for other components
of the HMC to bind and repress Hap1 in the absence of heme. We thought
that all these HRMs and the 17-amino-acid repeats would collectively
contribute to a high degree of heme regulation. If this model is
correct, the extent of Hap1 heme responsiveness would gradually
diminish with the progressive deletion of the HRMs and 17-amino-acid
repeats. We therefore examined the effect of deletion of the HRMs and
17-amino-acid repeats on Hap1 heme responsiveness. However, in vivo
analysis of Hap1 mutants defective in heme regulation is difficult
because of their toxicity under heme-deficient conditions (20,
33). None of the previously generated Hap1 deletion mutants was
examined under heme-deficient conditions (20, 33), so the
extent of heme responsiveness of these mutants could not be determined.
To overcome this problem, we expressed wild-type and Hap1 mutants from
the inducible GAL1-10 promoter, which is not heme regulated
(9). However, the inducible GAL1-10 promoter is a
strong promoter. The full induction of this promoter can lead to the
overexpression of the Hap1 proteins, which may minimize the effects of
mutations on heme regulation. We therefore examined the Hap1 proteins
at a low expression level, an intermediate expression level, and a high
expression level (27). The low expression level was achieved
by adding 0.25% galactose-1.75% glucose to the culture grown on
noninducing, nonrepressing raffinose medium. Under this condition, the
activity of the GAL1-10 promoter-lacZ reporter is
indistinguishable from the basal level (8, 12). Intermediate
and high expression levels were achieved by adding 1% galactose-1%
glucose and 2% galactose to the cultures, respectively
(27). Under the condition for the intermediate expression
level, the activity of the GAL1-10 promoter is about 5% of
that under the high (full) induction condition.
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FIG. 1.
Deletion analysis of functional modules in the heme
domain critical for heme regulation. (A) Structures of Hap1 derivatives
with deletions in the heme domain and their transcriptional activity in
the presence and absence of heme. Shown are the zinc cluster (residues
55 to 95), the dimerization domain (DD) (residues 117 to 244), the
previously designated heme domain (residues 245 to 444), HRM7 (near
residue 1192), and the activation domain (ACT) (residues 1309 to 1483).
In the heme domain, the lightly shaded box represents RPM1 (residues
245 to 278); the heavily shaded boxes represent the six heme-responsive
motifs; the open boxes represent three 17-amino-acid repeats; and the
angled lines represent the deleted regions. Residues 387 to 444, 345 to
444, 321 to 444, 278 to 444, 245 to 278, and 245 to 444 are deleted in
HH4, HH3, HH2, HH0, HH, and heme, respectively. MHY200 cells were
cotransformed with an expression vector producing wild-type Hap1 or
mutants and a UAS1-TATA-lacZ reporter. Cells were induced
with 0.25% galactose-1.75% glucose (low expression level), 1%
galactose-1% glucose (intermediate expression level), and 2%
galactose (high expression level), respectively. -Galactosidase
(-gal) activities (mean ± standard error) were detected in
heme-deficient cells (low [ALA]) and heme-sufficient cells (high
[ALA]). (B) Western blot showing the expression levels of
wild-type Hap1 and deletion mutants in yeast cells. Yeast extracts (150 µg) containing wild-type Hap1 (lane 1), HH4 (lane 2), HH3 (lane 3),
HH2 (lane 4), HH0 (lane 5), HH (lane 6), and heme (lane 7) were
analyzed on an SDS-7% polyacrylamide gel, transferred, and detected
by using an antibody against GST-Hap1. Extracts were prepared from
cells induced with 2% galactose.
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|
As expected, at all expression levels, Hap1 activity was low under
heme-deficient conditions (as described in the legend to
Fig.
1A). In
heme-sufficient cells, Hap1 activity was highest
at the intermediate
expression level, very likely because this
Hap1 level was high enough
to achieve maximum transcriptional
activity but not enough to cause
squelching (
7), which might
occur when Hap1 was expressed at
a high level. In fact, in heme-sufficient
cells, most mutants showed
the highest activity when they were
expressed at an intermediate level
(Fig.
1 and later figures).
Nevertheless, overexpressing wild-type Hap1
or mutants did not
significantly change the extent of heme
responsiveness (Fig.
1A
and later figures). Strikingly, progressive
deletion and even
complete deletion of the HRMs and 17-amino-acid
repeats did not
abolish Hap1 repression in the absence of heme (low
[
ALA]) (HH4
to HH0 in Fig.
1A), or Hap1 activation by heme (high
[
ALA]).
HH2 and HH0 were even more heme responsive: they were much
more
repressed than wild-type Hap1 but were activated by heme to nearly
the same level as wild-type Hap1. These results suggest that HRM1
to -6 and 17-amino-acid repeats in the heme domain are not responsible
for
heme
regulation.
However, deletion of residues 245 to 278 (HH in Fig.
1A) outside HRM1
to -6 and 17-amino-acid repeats had a drastic effect
on heme
regulation: at all expression levels, HH activity in the
absence of
heme was at least as high as that of wild-type Hap1
in the presence of
heme (compare HH and Hap1 in Fig.
1A), suggesting
that the mutant was
completely derepressed in the absence of heme.
Deletion of the whole
heme domain (
heme) also caused the complete
derepression of Hap1. It
is not clear why, at a low expression
level, the activity of
heme
was higher in the absence of heme
than in the presence of heme. In
fact, all derepressed deletion
mutants (Fig.
1; also see Fig.
2 and
3)
were barely stimulated
by heme at the low expression level. To rule out
the possibility
that different protein levels, caused by variations in
the stability
of mutants, contributed to the derepression of HH and
heme and
to varying transcriptional activities of other mutants, we
carried
out Western blotting analysis. The amounts of wild-type Hap1 or
mutants were detectable by Western blotting only at the high expression
level, but we expected the relative amounts to remain approximately
the
same at all expression levels. As shown in Fig.
1B, fusions
from HH4 to
HH0 were expressed at a level identical to wild-type
Hap1, suggesting
that the gradual decrease in the activity of
these mutants was not
attributable to the difference in protein
levels. Furthermore, the
constitutive mutants HH and
heme were
produced at a lower level than
that of wild-type Hap1 or other
mutants, suggesting that the high
transcriptional activity of
HH and
heme was not due to a high
protein level. These results
demonstrate that residues 245 to 278, not
the HRMs or 17-amino-acid
repeats, are responsible for Hap1 repression
in the absence of
heme. The region encompassing residues 245 to 278 is
named repression
module 1 (RPM1) (Fig.
1A) hereafter in this
work.
Functional analysis of the HRM7 domain shows that the module
encompassing residues 1061 to 1185, not HRM7, is critical for Hap1
repression in the absence of heme.
Previous studies identified an
HRM (HRM7) containing amino acid residue 1192. Mutations of amino acid
residues near HRM7 affect Hap1 heme responsiveness (10, 33),
suggesting that the domain encompassing HRM7 is important for heme
regulation of Hap1. However, this domain contains a large region with
very little informative sequence or few structural characteristics,
except for HRM7. We therefore determined which parts of this region
contribute to heme regulation of Hap1 by systematically testing various
deletions and mutations in this domain for their effects on Hap1
activity (Fig. 2). As shown in Fig. 2A,
mutants of the HRM7 domain were analyzed under low, intermediate, and
high expression levels (Fig. 2A). At the low expression level, H7-d1,
with residues 447 to 1309 deleted, and H7-d2, with residues 746 to 1309 deleted, were much less active than wild-type Hap1 or mutants with
smaller deletions, probably because these large deletions significantly
perturbed the protein structure (note that these proteins were stably
expressed as shown by Western analysis [Fig. 2B]). At the
intermediate expression level, these mutants showed a moderate level of
activity both in the absence and presence of heme, and heme stimulated
their activity about four- to sixfold (Fig. 2A). At the high expression level, these two mutants were derepressed in the absence of heme. They
showed an activity in the absence of heme higher than that of wild-type
Hap1 in the presence of heme (Fig. 2A). Heme hyperstimulated the
mutants' activity three- to fivefold. To pinpoint the residues critical for heme regulation, we deleted a smaller region, residues 1061 to 1309 (H7-d3). At all expression levels, H7-d3 was derepressed in the absence of heme. At the intermediate or high expression level,
H7-d3 was further stimulated about fourfold by heme. The results show
that the residues critical for Hap1 repression in the HRM7 domain are
located within the region encompassing residues 1061 to 1309.
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FIG. 2.
Deletion and mutational analyses of Hap1 modules in the
HRM7 domain. (A) Domain structures of wild-type Hap1 and mutants and
their activity in the presence and absence of heme. Shown here are the
zinc cluster (Zn), the dimerization domain (DD), RPM1, HRM1 to -6, RPM2
(residues 1161 to 1185), HRM7, and the activation domain (ACT). Deleted
regions are marked by dashed lines. In H7-1 and H7-2, the Cys residue
in the HRM7 motif is mutated to Ala. Cells were induced with 0.25%
galactose-1.75% glucose (low expression level), 1% galactose-1%
glucose (intermediate expression level), and 2% galactose (high
expression level), respectively. -Galactosidase (-gal) activities
were detected in heme-deficient cells (low [ALA]) and
heme-sufficient cells (high [ALA]). (B) Western blots showing the
expression levels of wild-type Hap1 and mutants in yeast cells.
Extracts (100 µg) containing wild-type Hap1 (lanes 1 and 8), H7-d3
(lane 2), H7-d5 (lane 3), H7-d7 (lane 4), H7-d6 (lane 5), H7-d8 (lane
6), and H7-d9 (lane 7); 50 µg of extracts containing H7-d1 (lane 9),
H7-d2 (lane 10), H7-d4 (lane 11), and H7-1 (lane 12); and 20 µg of
H7-2 (lane 13) were analyzed on SDS-7% polyacrylamide gels,
transferred, and detected by using an antibody against GST-Hap1. The
minor bands below the main band were due to Hap1 degradation. Extracts
were prepared from cells induced with 2% galactose.
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To narrow down the critical residues, mutants with smaller deletions
were generated. Deletion of residues 1061 to 1200 (H7-d5)
caused Hap1
derepression in the absence of heme, while deletion
of residues 1198 to
1309 (H7-d4) abolished Hap1 activity even
in the presence of heme (Fig.
2A). The region encompassing residues
1061 to 1200 was further divided
into two segments and deleted
separately. Deletion of residues 1061 to
1140 (H7-d7) (Fig.
2A)
caused Hap1 derepression in the absence of heme.
At intermediate
and high expression levels, the mutant was hyperinduced
by heme
by fourfold. Interestingly, deletion of residues 1141 to 1185
(H7-d6) (Fig.
2A), immediately adjacent to the HRM7 motif, also
caused
the full derepression of Hap1. These results show that
residues 1061 to
1140 and residues 1141 to 1185 are both critical
for Hap1 repression in
the absence of heme. We therefore defined
the region encompassing
residues 1061 to 1185 as RPM2. Residues
1061 to 1140 in RPM2 were
further divided into two parts, residues
1061 to 1100 (H7-d8) (Fig.
2A)
and residues 1101 to 1140 (H7-d9)
(Fig.
2A), and each part was deleted
separately. Notably, these
deletions did not cause Hap1 to be
derepressed, suggesting that
these parts provide redundant functions in
Hap1 repression. Western
blotting analysis showed that all mutants were
produced at levels
similar to the wild-type level (Fig.
2B). The small
variation
of protein levels among mutants did not correlate with the
variation
in the mutant activity (Fig.
2). For example, the
constitutive
mutants H7-d1, H7-d2, H7-d3, H7-d5, and H7-d7 were
expressed at
a slightly lower level than the totally inactive mutant
H7-d4
and the repressed mutants H7-d8 and H7-d9. Therefore, heme
responsiveness
of these mutants was not dictated by their expression
levels.
These experiments show that not the HRM7 motif but rather RPM2,
which contains residues 1061 to 1185, is critical for Hap1 repression
in the absence of heme. This module appears to be composed of
three
parts: residues 1061 to 1100, residues 1101 to 1140, and
residues 1141 to 1185. Residues 1141 to 1185 can cooperate with
either residues 1061 to 1100 (in H7-d9) or residues 1101 to 1140
(in H7-d8) to repress Hap1
in the absence of
heme.
Evidently, HRM7 is not responsible for Hap1 repression. To further
investigate the role of the HRM7 motif in heme regulation,
we generated
a mutant containing a Cys-to-Ala change at position
1193. This Cys
residue within the HRM7 motif is critical for heme
binding in vitro
(
33). As shown in Fig.
2A, mutating HRM7 (mutant
H7-1) did
not affect Hap1 repression in the absence of heme but
reduced its
activity in the presence of heme, at all expression
levels. The
reduction in heme activation was further reinforced
in the double
mutant H7-2 (Fig.
2A), with HRM1 to -6 deleted and
HRM7 mutated. Not
surprisingly, H7-2 showed an even lower activity
than H7-1 in the
presence of heme but remained repressed in the
absence of heme. The low
activity of H7-1 and H7-2 was not attributable
to low protein
expression levels, because Western blotting analysis
showed that these
mutants were produced at an even higher level
than that of wild-type
Hap1 (Fig.
2B). These results suggest that
the HRM7 motif has no role
in repressing Hap1 in the absence of
heme but plays a role in heme
activation of Hap1 or transcriptional
activation by Hap1 (see also
below).
A module encompassing residues 203 to 244 in the dimerization
domain is critical for Hap1 repression in the absence of heme.
Previous experiments suggest that the Hap1 dimerization domain is also
important for heme regulation (35). However, it is not clear
from these experiments whether the entire dimerization domain or just a
segment of the domain is important for heme regulation. We therefore
divided the dimerization domain into four segments and determined
whether deletion of each segment affects Hap1 heme responsiveness.
These segments contain residues 203 to 244, 172 to 202, 136 to 171, and
105 to 135, respectively. As shown in Fig.
3A, deletion of residues 203 to 244 (mutant DD1) caused the complete derepression of Hap1. At all
expression levels, the activity of DD1 in the absence of heme was
nearly as high as or higher than that of wild-type Hap1 in the presence
of heme, even though the protein level of DD1 was much lower than that
of wild-type Hap1 (Fig. 3). Deletion of residues 172 to 202 (DD2) or
136 to 171 (DD3) did not considerably affect Hap1 repression or
activation by heme. Deletion of residues 105 to 135 (DD4) caused Hap1
activity to be lower, both in the presence and absence of heme. Because heme significantly stimulated the activity of DD4 at the intermediate (14-fold) or high (41-fold) expression level, the low activity in
heme-sufficient cells was very likely caused by the low transcriptional activity of the mutant, not by deficiency in heme activation. Again,
Western blotting analysis showed that all deletion mutants except for
the derepressed mutant DD1 were produced at a level similar to the
wild-type Hap1 level (Fig. 3B). These results show that the segment
containing residues 203 to 244, termed RPM3 hereafter in this work, is
critical for Hap1 repression.
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FIG. 3.
Deletion and mutational analyses of the Hap1
dimerization domain. (A) Hap1 mutants with deletions in the
dimerization domain and their activity in the presence and absence of
heme. Shown here are the zinc cluster (Zn), the dimerization domain
(DD), RPM3 and RPM1 (RPM3/1), RPM2, HRM1 to -6, HRM7, and the
activation domain (ACT). In DD1 to DD4, DH1, and DH2, residues 203 to
244, 172 to 202, 136 to 171, 105 to 135, 203 to 278, and 225 to 257 are
deleted. MHY200 cells were cotransformed with an expression vector
producing wild-type Hap1 or mutants and a UAS1-TATA-lacZ
reporter. Cells were induced with 0.25% galactose-1.75% glucose (low
expression level), 1% galactose-1% glucose (intermediate expression
level), and 2% galactose (high expression level), respectively.
-Galactosidase (-gal) activities (mean ± standard error) of
wild-type Hap1 and mutants were detected in heme-deficient cells (low
[ALA]) and heme-sufficient cells (high [ALA]). The mutations
in the mutants M-2H and M-H4 are shown. The primary amino acid sequence
of Hap1 residues 201 to 280 is SSSLSISNKY DNDELDLTKD
FDLLHIKSNG TIHLGATHWL SIMKGDPYLK
LLWGHIFAMR EKLNEWYYQK NSYSKLKSSK (boldface type
indicates the four His residues). (B) Western blot showing the
expression levels of wild-type Hap1 and mutants in yeast cells.
Extracts (100 µg) containing DD2 (lane 2) and DD3 (lane 3) and 200 µg of extracts containing wild-type Hap1 (lane 5), DD1 (lane 1), DD4
(lane 4), DH1 (lane 6), DH2 (lane 7), M-2H (lane 8), and M-H4 (lane 9)
were analyzed on an SDS-7% polyacrylamide gel, transferred, and
detected by using an antibody against GST-Hap1. Extracts were prepared
from cells induced with 2% galactose. The lower bands below the major
band were due to degraded Hap1 fragments.
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RPM3, which contains residues 203 to 244, is contiguous to RPM1, which
contains residues 245 to 278 (Fig.
3A). These two modules
may closely
cooperate to mediate heme regulation. Inspection of
the sequences in
these regions revealed four His residues: His
225, His 233, His 238, and His 255 (as described in the legend
to Fig.
3). Because His
residues are often involved in heme binding
in hemoglobin and certain
cytochromes, we speculated that these
four residues might contribute to
heme binding and, hence, heme
regulation. If so, then deletion of the
region containing these
four His residues would abolish heme
regulation. As shown in Fig.
3A, deletion of residues 225 to 257 (DH2),
like deletion of residues
203 to 278 containing both RPM1 and RPM2
(DH1), caused Hap1 derepression
at all expression levels, although the
protein levels of these
mutants appeared to be lower than that of
wild-type Hap1 (Fig.
3B). DH2 had a slightly higher activity, both in
the presence
and absence of heme, than did DD1 and DH1, probably
because its
protein level was slightly higher (Fig.
3B). These results
show
that the region containing the four His residues is indeed
critical
for Hap1 repression in the absence of heme. To explore further
the possibility that the four His residues are critical for heme
regulation, we generated two mutants: mutant M-2H with His 233
and His
238 changed to Ala and mutant M-H4 with His 255 changed
to Thr. Mutant
M-2H was partially derepressed in the absence of
heme and further
stimulated four- to eightfold by heme, at all
expression levels (Fig.
3A). Mutation of the His 255 to Thr in
mutant M-H4 had no effect on
Hap1 repression in the absence of
heme, but its activity was lower in
the presence of heme, probably
because its protein level was lower
(Fig.
3B). These results suggest
that His 233 and His 238 are important
but are not the only residues
responsible for heme
regulation.
Some derepressed mutants are defective in forming HMC in the
absence of heme.
Our in vivo functional analysis of Hap1 mutants
identified three new repression modules, RPM1 to -3, that are important
for Hap1 repression in the absence of heme. Previous experiments
suggest that a higher-order complex, termed HMC, plays an important
role in Hap1 repression (6, 32, 35). If RPMs mediate Hap1
repression by permitting HMC formation, then derepressed mutants would
be defective in HMC formation. Therefore, we determined whether the mutants described in Fig. 1 to 3 can form the HMC. Previously, it was
shown that in the absence of heme, Hap1 forms the HMC even when its
expression level is low (but high enough for detecting the HMC)
(6, 32). As the Hap1 concentration increases, the amount of
the HMC gradually increases and reaches the highest level when non-Hap1
proteins in the HMC become limiting. Excess Hap1 forms the dimeric
complex even in the absence of heme (6, 32). Under the
conditions for the low and intermediate expression levels used for
-galactosidase assays, the amounts of wild-type or mutant Hap1
proteins were not sufficient for the detection of the HMC or the
dimeric complex (27). We therefore expressed various amounts
of wild-type and mutant Hap1 proteins by varying the induction times,
as previously described (Fig. 4A)
(6, 32). As shown in previous studies (6, 32) and
in Fig. 4A, when the Hap1 amount was low, the HMC, not the dimeric
complex, was formed in the absence of heme (Fig. 4A, lane 1 [only
three different amounts are shown in Fig. 4A for brevity]). Heme
disrupted the HMC and permitted Hap1 to bind to DNA with much higher
affinity (lane 2). As the Hap1 amount increased, more HMC formed (lanes 3 and 5). The dimeric complex also formed in the absence of heme at the
highest Hap1 level (lane 5).
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FIG. 4.
The effect of protein expression levels on the formation
of the HMC. (A) Formation of the HMC and the dimeric complex by
increasing amounts of wild-type Hap1. Yeast cells bearing the
expression plasmid for wild-type Hap1 under the control of the
GAL1-10 promoter grown in 2% raffinose were induced with
2% galactose for 1 h (lanes 1 and 2), 4 h (lanes 3 and 4),
and 8 h (lanes 5 and 6). Extracts were prepared from these cells
and analyzed in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of heme by DNA mobility shift assays on 3.5% polyacrylamide
gels. (B) Formation of the HMC and dimeric complex by increasing
amounts of H7-d8. Yeast cells bearing the expression plasmid for H7-d8
under the control of the GAL1-10 promoter grown in 2%
raffinose were induced with 2% galactose for 1 h (lanes 1 and 2),
2 h (lanes 3 and 4), and 4 h (lanes 5 and 6). Extracts were
prepared from these cells and analyzed in the absence (lanes 1, 3, and
5) or presence (lanes 2, 4, and 6) of heme by DNA mobility shift
assays. Note that H7-d8, like wild-type Hap1, also formed the dimeric
complex in the absence of heme when extracts were prepared from cells
induced with galactose for longer periods (Fig. 5B). (C) Formation of
the HMC and dimeric complex by increasing amounts of heme. Yeast
cells bearing the expression plasmid for heme under the control of
the GAL1-10 promoter grown in 2% raffinose were induced
with 2% galactose for 1 h (lanes 1 and 2), 2 h (lanes 3 and
4), and 4 h (lanes 5 and 6). Extracts were prepared from these
cells and analyzed in the absence (lanes 1, 3, and 5) or presence
(lanes 2, 4, and 6) of heme by DNA mobility shift assays. (D) Formation
of the HMC and dimeric complex by increasing amounts of H7-d6. Yeast
cells bearing the expression plasmid for H7-d6 under the control of the
GAL1-10 promoter grown in 2% raffinose were induced with
2% galactose for 1 h (lanes 1 and 2), 2 h (lanes 3 and 4),
and 4 h (lanes 5 and 6). The band below the dimeric complex was
very likely due to degradation of the mutant protein. Extracts were
prepared from these cells and analyzed in the absence (lanes 1, 3, and
5) or presence (lanes 2, 4, and 6) of heme by DNA mobility shift
assays.
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If HMC formation is required for Hap1 repression in the absence of
heme, then repressed mutants would form the HMC at the
same level as
wild-type Hap1, whereas derepressed mutants would
have reduced levels
of HMC. Indeed, as shown in Fig.
4, the repressed
mutant H7-d8, like
wild-type Hap1, formed more HMC as its amount
increased (Fig.
4B) (note
that H7-d8 was not overexpressed enough
to form the dimeric complex in
the absence of heme; after a longer
induction period, as shown in Fig.
5B, H7-d8 did form the dimeric
complex). However, unlike wild-type Hap1
and H7-d8, the
heme
mutant, which was derepressed approximately
100-fold relative
to wild type Hap1 (Fig.
1A), formed very little HMC
(about 2%
of that formed by wild-type Hap1) (compare lanes 1, 3, and 5 in
Fig.
4C to those in Fig.
4A). Instead, even in the absence of
heme,
a significant amount of the dimeric complex was formed at
all
expression levels. Similarly, another derepressed mutant,
H7-d6, which
was derepressed approximately 20- to 50-fold relative
to wild-type Hap1
(Fig.
2A), formed significantly less HMC (about
10% that of wild-type
formed by Hap1) at all expression levels
(compare Fig.
4D to
4A). H7-d6
also formed the dimeric complex
in the absence of heme, even when its
amount was low (Fig.
4D,
lanes 1 and 2). These titration experiments
show that in the absence
of heme, wild-type Hap1 and the repressed
H7-d8 mutant preferentially
formed the HMC whereas the derepressed
heme and H7-d6 mutants
preferentially formed the dimeric
complex.
These and previous experiments (
6,
32) show that
overexpressing Hap1 does not interfere with HMC formation but optimizes
the sensitivity of detection of the HMC. Therefore, to determine
whether other mutants can form the HMC, we analyzed extracts prepared
from cells expressing high levels of mutants. Although this approach
cannot detect subtle differences in the HMC, it provides the optimum
conditions for determining whether a mutant can form the HMC.
If a
mutant cannot form the HMC when overexpressed, it is unlikely
to form
the HMC when expressed at lower levels. As shown in Fig.
5, several derepressed mutants
HH and
heme (Fig.
5A); H7-d3,
H7-d5, and H7-d7 (Fig.
5B); and DD1 and DH1
(Fig.
5C)
did not
form a discernible amount of the HMC under the test
condition
(the signal at the position of the HMC band for the mutants
was
less than 5% of that for wild-type Hap1). Instead, they formed
the
dimeric complex and bound strongly to DNA in the absence of
heme. Heme
did not stimulate DNA binding by these mutants; it
actually weakened
DNA binding by HH and
heme (Fig.
5A). DNA binding
by DD1 was
stimulated slightly by heme, but no discernible HMC
band was detected
(Fig.
5C, lane 8). (H7-d1 and H7-d2 are not
shown here because no
distinctive bands were observed when extracts
containing these mutants
were analyzed, whether or not heme was
present.) In sum, eight mutants,
seven shown in Fig.
5 and H7-d6
shown in Fig.
4D, appeared to
preferentially form the dimeric
complex rather than the HMC. This
suggests that they are defective
or partially defective in HMC
formation, although it is still
possible that they can form the HMC if
their protein levels in
extracts are made as high as that of wild-type
Hap1 (the protein
levels of derepressed mutants in extracts were
usually lower than
that of wild-type Hap1 [Fig.
1B,
2B, and
3B]). One
derepressed
mutant (DH2) (Fig.
5C) formed a stable HMC. All repressed
mutants
formed the HMC (see mutants HH4 to HH0 in Fig.
5A; mutants
H7-d4,
H7-d8, H7-d9, H7-1, and H7-2 in Fig.
5B; and mutant DD2 in Fig.
5C; other mutants of the dimerization domain behaved identically
to DD2
[not shown]). The intensity of HMCs formed by these mutants
was about
the same (70 to 120%) as that formed by wild-type Hap1.
These results
suggest that repressed mutants always retain the
ability to form the
HMC, while many but not all derepressed mutants
are defective in
forming the HMC.
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FIG. 5.
Analysis of DNA-binding complexes formed by wild-type
Hap1 and mutants. (A) DNA-binding complexes formed by deletion mutants
in the heme domain. Extracts containing wild-type Hap1 (lanes 1 and 2),
HH4 (lanes 3 and 4), HH3 (lanes 5 and 6), HH2 (lanes 7 and 8), HH0
(lanes 9 and 10), HH (lanes 11 and 12), and heme (lanes 13 and 14)
were incubated with radiolabeled DNA in the absence (lanes 1, 3, 5, 7, 9, 11, and 13) or presence (lanes 2, 4, 6, 8, 10, 12, and 14) of heme.
The reaction mixtures were analyzed on a 3.5% polyacrylamide gel. (B)
DNA-binding complexes formed by deletion mutants in the HRM7 domain.
Extracts containing Hap1 (lanes 1 and 2), H7-d4 (lanes 3 and 4), H7-d8
(lanes 5 and 6), H7-d9 (lanes 7 and 8), H7-1 (lanes 9 and 10), H7-2
(lanes 11 and 12), H7-d3 (lanes 13 and 14), H7-d5 (lanes 15 and 16),
and H7-d7 (lanes 17 and 18) were incubated with radiolabeled DNA in the
absence (lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17) or presence (lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18) of heme. H7-d1 and H7-d2 are not shown
here because no distinctive bands were observed when they were
subjected to DNA mobility shift assays. (C) DNA-binding complexes
formed by deletion mutants in the dimerization domain. Extracts
containing DH2 (lanes 1 and 2), DH1 (lanes 3 and 4), DD2 (lanes 5 and
6), DD1 (lanes 7 and 8), and wild-type Hap1 (lanes 9 and 10) were
incubated with radiolabeled DNA in the presence (lanes 1, 3, 5, 7, and
9) or absence (lanes 2, 4, 6, 8, and 10) of heme. DD3 and DD4 behaved
the same as DD2 and are not shown here. The positions of the HMC and
dimeric complex (DC) are marked. All extracts used here were prepared
from JEL1 cells bearing expression plasmids induced with 2% galactose
for 8 h.
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A higher heme concentration is required to disrupt the HMC when the
HRM7 motif is mutated.
Mutants H7-1 and H7-2 had a much lower
activity than wild-type Hap1 even in heme-sufficient cells, but these
mutants were expressed at an even higher level than wild-type Hap1
(Fig. 2B). Thus, the reduced activity most likely results from
hyperrepression caused by an HMC insensitive to heme or the loss of
transcription-activating activity of the activation domain. Since the
activation domain usually acts independently of other domains and since
mutating amino acid residues to Ala often does not cause drastic
changes in protein conformation (29), it is unlikely that
the mutations in H7-1 and H7-2 weakened the activation domain. However,
HRMs can bind to heme directly in vitro (33), and Fig. 5B
reveals that the HMC formed by H7-2 was not totally disrupted by heme (lane 12). Therefore, it is conceivable that H7-1 and H7-2 might be
defective in heme binding, thereby forming HMCs less sensitive to heme
and, therefore, hyperrepressed. If this line of reasoning is correct,
then H7-1, which was more repressed than wild-type Hap1 (Fig. 2), would
form an HMC that is more resistant to heme than the HMC formed by
wild-type Hap1. Likewise, H7-2, which was more repressed than H7-1,
would form an HMC that is even more resistant to heme than the HMC
formed by H7-1. This is indeed the case as shown in Fig.
6. The HMC formed by wild-type Hap1 required only 0.1 to 0.5 ng of heme per µl for dissociation (Fig. 6,
lanes 1 to 6), and H7-1 required 0.5 to 2 ng/µl (Fig. 6, lanes 7 to
12), while H7-2 required 2 to 10 ng/µl for dissociation (Fig. 6,
lanes 13 to 18). These results suggest that H7-1 forms a more stable
HMC than wild-type Hap1 and H7-2 forms an even more stable HMC than
H7-1. We also attempted to determine whether the activity of H7-1 and
H7-2 can be enhanced by the addition of high levels of the heme
precursor, deuteroporphyrin IX, or ALA. However, we did not observe
any significant increase under high concentrations of deuteroporphyrin
IX or ALA, very likely because heme concentration in vivo cannot
reach the same level as in vitro due to the characteristics of enzymes
involved in heme synthesis. Nonetheless, the data on H7-1 and H7-2
shown in Fig. 2 and 6, together with the previous finding that a
peptide containing an HRM can bind heme directly, strongly support the
idea that HRMs can bind to heme and mediate heme activation of Hap1 in
vivo.
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FIG. 6.
Effect of various heme concentrations on the HMCs formed
by wild-type Hap1, H7-1, and H7-2. Extracts containing wild-type Hap1
(lanes 1 to 6), H7-1 (lanes 7 to 12), and H7-2 (lanes 13 to 18) were
incubated with radiolabeled DNA under the following heme concentrations
(in nanograms per microliter): 0 (lanes 1, 7, and 13), 0.02 (lanes 2, 8, and 14), 0.1 (lanes 3, 9, and 15), 0.5 (lanes 4, 10, and 16), 2 (lanes 5, 11, and 17) and 10 (lanes 6, 12, and 18). The reaction
mixtures were analyzed on a 3.5% polyacrylamide gel. The positions of
the HMC and dimeric complex (DC) are marked.
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DISCUSSION |
This report provides significant new insights into the mechanism
by which Hap1 is repressed in the absence of heme and is subsequently
activated by heme. Three themes emerge from the analyses of various
Hap1 mutants. First, three novel repression modules, RPM1 to -3, located in the previously designated dimerization domain, the heme
domain, and the HRM7 domain, are responsible for Hap1 repression in the
absence of heme. Second, the repression modules RPM1 to -3 mediate Hap1
repression, at least in part, through the formation of the HMC. Third,
HRMs, in particular HRM7, mediate heme activation of Hap1, evidently by
permitting heme binding and the disassembly of the HMC. These themes
are further discussed below.
Novel modules critical for Hap1 repression in the absence of
heme.
Three novel repression modules, RPM1 to -3, not the
previously identified HRMs, are critical for Hap1 repression in the
absence of heme. The first module, RPM1 (Fig. 1), lies within residues 245 to 278 in the previously designated heme domain. The second module,
RPM2 (Fig. 2), lies within residues 1061 to 1185 near the HRM7 motif.
The third module, RPM3 (Fig. 3A), lies within the previously designated
dimerization domain and encompasses residues 203 to 244. RPM1 to -3 act
synergistically to repress Hap1 activity in the absence of heme because
deletion of any one of these modules caused Hap1 derepression in the
absence of heme (see Fig. 1 to 3). Intriguingly, the data show that the
distinctive HRM1 to -6 and the three 17-amino-acid repeats in the
previously designated heme domain have no effect on Hap1 repression in
the absence of heme, although HRM1 to -6 appear to play a minor role in
heme activation (see below).
Possible mechanisms by which RPMs mediate Hap1 repression.
Previous experiments linked HMC formation to Hap1 repression
(35). The data here show that RPM1 to -3 are essential for Hap1 repression. We imagine two possible mechanisms by which RPMs can
confer Hap1 repression. One possibility is that RPMs interact with Hap1
DNA-binding and/or activation domains, thereby masking and repressing
the DNA-binding and/or activation domains by intramolecular interactions. Deletion of any RPM would therefore disrupt the interactions and result in unmasking of these domains, leading to Hap1
derepression. Alternatively, RPM1 to -3 may be involved in HMC
formation and thus repress Hap1. Deletion of any RPM would then disrupt
the HMC, causing Hap1 derepression. The data suggest that RPMs are at
least partially involved in forming the HMC (see below), but do not
rule out the possibility that intramolecular interactions are important
for Hap1 repression. These two possibilities are not mutually
exclusive. Perhaps both intramolecular interactions and HMC formation
play important roles in Hap1 repression.
Previous reports (
6,
32) and data shown in Fig.
4 show that
in the absence of heme, as the amount of wild-type Hap1 and
H7-d8
increased, the HMC was formed first, followed by the dimeric
complex,
which formed when the HMC became saturated. Unlike wild-type
Hap1 and
H7-d8, 8 of 11 derepressed mutants
including HH and
heme
(Fig.
5A);
H7-d3, H7-d5, and H7-d7 (Fig.
5B); H7-d6 (Fig.
4D);
and DH1 and DD1
(Fig.
5C)
preferentially formed the dimeric complex,
whether or not
the HMC was observed. Therefore, we conclude that
the derepressed
mutants are defective in forming the HMC. One
argument against this
idea is that the protein levels of many
derepressed mutants may be
lower than those of wild-type Hap1
and repressed mutants. Thus, the
potential HMCs formed by derepressed
mutants could be more difficult to
detect. However, in the absence
of heme, the derepressed mutants formed
a significant amount of
dimeric complex (Fig.
4C and D and 5). For
wild-type Hap1 (
6,
32) and the repressed mutant H7-d8 (Fig.
4A and B and 5C), this
larger amount of dimeric complex was formed only
when they were
overexpressed and when the HMC became saturated.
Although these
derepressed mutants may still form the HMC if the
protein levels
were higher, they appeared to preferentially form the
dimeric
complex and were at least partially defective in forming the
HMC.
Another argument is that the derepressed mutants may form
defective
HMCs that cannot bind to DNA, making their detection
difficult.
Our data cannot totally exclude this possibility, but we do
not
think it is likely for the following reasons. First, many
derepressed
mutants that showed reduced dimer binding also had a lower
protein
level (HH and
heme [Fig.
1B and
5A], H7-d5 and H7-d7
[Fig.
2B
and
5B], and DH1 and DD1 [Fig.
3B and
5C]). Therefore, the
reduced
dimer binding was very likely due to reduced protein levels,
not
due to reduced affinity. Second, previous and recent data show
that
Hap1 mutations similarly affect HMC binding and Hap1 dimer
binding and
that the HMC and Hap1 dimer recognize various DNA
sites in the same
manner (
11,
32). It is therefore unlikely
that mutations in
the derepressed mutants selectively affected
HMC binding but not dimer
binding. On the basis of these arguments,
the conclusion that these
derepressed mutants are at least partially
defective in forming the HMC
appears to be plausible. This deficiency
may be in part responsible for
the loss of repression of these
mutants. It is not clear why DH2 (Fig.
5C) is derepressed even
though it forms the HMC. Perhaps the HMC formed
by DH2 adopts
an altered conformation so that Hap1 can activate
transcription
even when associated with other proteins. This occurs in
the Gal4-Gal80
regulatory system in which activated Gal4 is still
associated
with its repressor Gal80 (
1,
15,
19,
22).
Alternatively,
perhaps the DNA-binding and activation domains of DH2
are totally
unmasked even though the HMC is
intact.
Regardless of whether RPMs act by permitting HMC formation, the data
clearly show that the three RPMs act cooperatively in
repressing Hap1.
Interestingly, protein structure analysis indicates
that residues 248 to 267 in RPM1 have a high potential of forming
an
-helix. The
helical-wheel representation of this module indicates
that most of the
hydrophobic residues are clustered on one face
of the helix. This
hydrophobic surface may interact with other
proteins in the HMC or the
Hap1 DNA-binding and activation domains,
thereby repressing Hap1. In
addition, residues 1089 to 1111 and
residues 1110 to 1134 in RPM2 have
a strong tendency to form an
-helix. These structural modules may
provide important intramolecular
or intermolecular interactions
critical for repressing
Hap1.
Role of HRMs in heme regulation.
Our data show that HRMs have
no impact on Hap1 repression in the absence of heme (Fig. 1 and 2).
Rather, the HRMs, in particular HRM7, are important for mediating heme
activation. HRM7 appeared to play a predominant role in mediating heme
activation. HRM7 alone (HH0 in Fig. 1A), but not HRM1 to -6 (H7-1 in
Fig. 2A), was sufficient for mediating efficient activation of Hap1 by
heme. HRM1 to -6 appeared to play an auxiliary role in mediating heme activation, which was revealed only when HRM7 was mutated in H7-2 (compare HH0 with wild-type Hap1 in Fig. 1A and H7-2 with H7-1 in Fig.
2A). The analysis of HMCs formed by H7-1 and H7-2 (Fig. 6) further
suggests that HRMs mediate heme activation by allowing heme binding and
subsequent disassembly of the HMC. H7-1 lacks a functional HRM7, so it
would bind to heme less efficiently than wild-type Hap1. Consequently,
the mutant HMC required a higher heme concentration to be disrupted
(Fig. 6), leading to hyperrepression in vivo (Fig. 2A). H7-2 lacks both
HRM7 and the six HRMs in the heme domain, so it would bind to heme even
less efficiently than H7-1. As a result, the mutant HMC required an
even higher heme concentration to be disrupted (Fig. 6) and was even
more repressed than H7-1 (Fig. 2A). HRMs do not appear to be the only
element responsible for heme binding and activation, because the mutant H7-2 was still stimulated by heme (Fig. 2A). Perhaps other residues in
Hap1 also bind to heme and potentiate Hap1 activation, or perhaps this
effect is mediated through heme binding to other proteins in the HMC.
Nevertheless, the data shown here strongly suggest that HRMs, in
particular HRM7, play an important role in heme binding and in
mediating heme activation of Hap1. Further, the data show that not all
HRMs are important for heme action although they all have the
capability to bind to heme in vitro (33). In the case of
Hap1, it is very surprising that deletion of all six HRMs in the heme
domain did not diminish Hap1 heme responsiveness (Fig. 1). This
surprising finding shows that the functional importance of HRMs in
other hemoproteins cannot be inferred from their mere presence but must
be determined by functional analysis. This explains why mutating the
HRMs in heme lyase and ALAS affects their functions while mutating the
HRMs in heme oxygenase and NF-E2 has no effect on the protein functions
(14, 16, 17, 26).
New model for heme regulation of Hap1.
Our studies provide a
new model for heme regulation of Hap1. The data show that two distinct
classes of elementsthe repression modules RPM1 to -3 and HRMsare
responsible for heme regulation of Hap1. The functions of both RPMs and
HRMs are linked to the HMC. RPMs appear to permit the formation of the
HMC, thereby allowing Hap1 to be repressed in the absence of heme.
Therefore, deletion of the RPMs can cause disruption or partial
disruption of the HMC, leading to Hap1 derepression (Fig. 1 to 5). HRMs
appear to permit heme binding and the disassembly of the HMC, thereby
leading to Hap1 activation by heme. Therefore, mutating HRMs causes the formation of mutant HMCs insensitive to heme and hyperrepression of the
mutants (see H7-1 and H7-2 in Fig. 2 and 6).
These studies on Hap1 heme regulation and transcriptional activation
may help reveal how the activities of other heme regulatory
proteins
are regulated. For example, modules in addition to HRMs
may play
important roles in mediating heme action in other hemoproteins
including HRI kinase, heme oxygenase, and NF-E2 (
3,
14,
16,
17,
26). In particular, HRI kinase binds to Hsp90 and Hsp70,
and this
interaction appears to be important for the regulation
of HRI kinase
activity (
3,
28). Perhaps similar modules in
Hap1 and HRI
kinase interact with these molecular chaperones and
mediate heme
regulation. The analyses here allow us to make two
general inferences
on the precise and sophisticated mechanisms
by which the activity of
many regulators can be controlled. First,
multiple modules in distant
regions of one protein can act together
to confer tight regulation. All
three RPMs of Hap1 together mediate
a high level of regulation, whereas
each alone can mediate only
a few fold of regulation (Fig.
1 to
3).
Multiple modules may also
be involved in the regulation of numerous
other transcription
factors, even though these modules may not be
important for DNA
binding or transcription-activating activity. Second,
regulation
of a transcription factor can be partitioned into repression
and
activation. Distinct classes of modules may be required for each
function. This may be a general mechanism by which the activity
of a
regulator can be controlled by different signals in a precise
and
stringent
manner.
| |
ACKNOWLEDGMENTS |
We thank W. Jelinek, H. Salomon, J. Qin, D. Conrad, J. Borowiec,
and H. Lee for critical reviewing and proofreading of the manuscript.
This work was supported by grants from the National Institutes of
Health (GM53453) and the National Science Foundation (MCB-96174720) to
L.Z.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, NYU Medical Center, 550 First Ave., New York, NY 10016. Phone: (212) 263-8506. Fax: (212) 263-8166. E-mail:
zhang102{at}mcrcr0.med.nyu.edu.
| |
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Abstract
Introduction
