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(Received for publication, November 1, 1994; and in revised form, December 27, 1994) From the
The Saccharomyces cerevisiae hemoglobin is a
flavoprotein of unknown function. It shares extensive sequence homology
with the globin of Candida as well as those of several
bacterial species. We have studied its gene regulation in order to
better understand its purpose in the cell. Transcriptional analyses
indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S.
cerevisiae globin message is induced during logarithmic growth and
under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription
factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to
intracellular heme levels. Anaerobically, there is a low level,
HAP-independent induction of hemoglobin mRNA. Unlike other systems
influenced by the HAP2/3/4 transcription factor complex, no activation
of hemoglobin expression by growth in non-fermentable carbon sources is
observed. Flavohemoglobin gene disruption does not alter cell viability
or growth in a variety of oxygen conditions and carbon sources.
Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the
formyltetrahydrofolate synthase (ADE3) locus. These data
indicate that, despite the high degree of homology, the S.
cerevisiae globin may have a function distinct from those proposed
for bacterial globins.
Hemoglobins have been detected spectrally and their genes cloned
from organisms representing all kingdoms of life, indicating a
primordial origin and a continuing widespread requirement for this
protein(1, 2, 3) . Although the role of
vertebrate hemoglobins as facilitators of oxygen diffusion is well
established, the function of globins in invertebrate animals, as well
as in plants, protozoa, fungi, and bacteria, is generally unclear.
Clues to the function of the microorganismal globins have come through
heterologous expression of hemoglobin from the aerobic bacteria Vitreoscilla. Overproduction of the Vitreoscilla globin (VtHb) ( Gene regulation studies of the
bacterial globins suggest a correlation in the functions of these
proteins. Hemoglobin levels increase over 50-fold in Vitreoscilla as cell density increases and oxygen tension drops(11) .
There is also an increase in reductase levels under the same
conditions, suggesting coordinate expression of the hemoglobin and
reductase activities in Vitreoscilla(10) . Remarkably,
the oxygen regulation of Vitreoscilla hemoglobin can also be
seen using the native VtHb promoter expressed in E. coli(12, 13) . Transcriptional fusions to the
hemoglobin promoter show a 5-7-fold increase in reporter gene
expression when cells are shifted to microaerophilic conditions or when
cells reach the end of log-phase growth. Limited oxygen supply also
causes a 20-fold increase in A. eutrophus globin
levels(14) . On the basis of promoter sequence homology, the
FNR and NARL transcription factors, which control anaerobic gene
expression in E. coli, have been proposed to affect expression
of the A. eutrophus globin(9) . A putative binding
site for FNR is also found in the promoter of the Vitreoscilla gene, and E. coli mutants lacking the FNR gene product do
not show microaerophilic activation of heterologous VtHb
transcription(15) . In S. cerevisiae, transcription
factors involved in oxygen regulation of gene expression have been well
characterized(16, 17, 18) . Heme, the
biosynthesis of which requires molecular oxygen in eukaryotes (19) , is an effector molecule involved in the sensing of
intracellular oxygen levels. For aerobically induced genes, HAP1 and
the HAP2/3/4 complex activate transcription in the presence of
heme(16) . Heme can also inhibit the transcription of hypoxic
or anaerobically expressed genes through the ROX1 and ORD1
transcription factors(20, 21) . To investigate
possible functions for the S. cerevisiae flavohemoglobin, we
have initiated gene regulation and disruption studies. Our results
indicate that S. cerevisiae hemoglobin expression is regulated
by cell density and oxygen in a manner different from that of the
bacterial globins, suggesting a separate and as yet undefined role for
this protein in aerobic yeast metabolism.
The yeast hemoglobin
reporter gene construct (pYH10) was generated by subcloning a SpeI-NheI restriction fragment containing
Figure 1:
Repression of YHG mRNA with increasing
cell density. Shown is the growth of strain YM4134 as a function of
time after an initial inoculation of 10
Figure 2:
YHG gene expression at varied oxygen
tensions. YM4134 was grown in YPGal at the defined oxygen
concentrations shown above the figure. Oxygen tensions were
maintained by bubbling defined gas mixtures continuously through the
cultures. All cultures also contained 10 µg/ml ergosterol and 0.1%
Tween 80 to allow anaerobic growth. After midlog phase was reached,
cells were chilled on ice with continued bubbling for 20 min, and RNA
was isolated. The blot was hybridized with a mixture of YHG and TIF51B
probes prepared as described under ``Materials and Methods.''
TIF51A is the 90% identical aerobic gene partner of TIF51B, which
cross-hybridizes to TIF51B probe. This blot was stripped and
re-hybridized with a S. cerevisiae ACT1 probe to ensure equal
loading of mRNA. Densitometry readings of the YHG message gave the
following intensities (setting 21% as 1.0): 1.26, 1.0, 0.50, 0.28,
0.23, 0.32, and 0.23.
Given the oxygen
responsiveness of the YHG gene, we examined YHG mRNA levels in a S.
cerevisiae strain (nem1) deleted in
Figure 3:
Effect of heme and heme-activated
transcription factors on YHG mRNA levels. RNA was isolated from midlog
phase cells grown in YPGal. The RNA blots were hybridized with the YHG
PCR probe. All cultures were supplemented with 10 µg/ml ergosterol
and 0.1% Tween 80. A, lanes show wild-type strain RZ53-6 (wt), RZ53-6hem1 (hem1), and
RZ53-6hem1 supplemented with 100 µg/ml
Strains deficient in HAP1
or HAP2 also display a decrease in YHG message (2.2- and 3.4-fold by
densitometry) compared with that of wild type (Fig. 3B, top). In a strain where both HAP1 and HAP2 have been mutated,
YHG mRNA levels drop about 50-fold from wild type, suggesting that the
heme/HAP pathway is predominant in the aerobic transcription of YHG. A
strain deficient in the heme-induced repressor ROX1 (RZ53-6 rox1) had no change in aerobic YHG gene expression (not
shown). The sharp decrease of transcription in the hap1/hap2 strain was surprising, given that there is some expression of YHG
even under anaerobic conditions (Fig. 2). To determine if the
anaerobic expression of YHG is HAP-independent, we isolated RNA from
wild-type cells and from the HAP mutants grown anaerobically. The
results, shown in the bottom of Fig. 3B,
reveal persistent anaerobic production of YHG mRNA even in the hap1/hap2 double mutant. To confirm that oxygen/heme
control of YHG mRNA is at the level of transcription, 1.6 kb of the YHG
promoter was fused to the lacZ reporter gene and integrated
into the genome of wild-type, hem1, hap1, hap
2, and hap1/hap2 strains, and lacZ assays were
performed (Table 2). A 4-fold decrease in lacZ activity
was observed in heme-deficient cells compared with wild-type levels.
Strains carrying a mutation in either HAP1 or HAP2 also showed about a
4-fold decrease from wild-type lacZ levels, whereas the hap1/hap2 double mutant produced about one-tenth of the
Figure 4:
Effect of culture density on expression of
the YHG promoter/lacZ construct. YM4134 carrying the
integrated pYH10 construct was grown in YPGal media and assayed for lacZ activities at the optical densities shown. The lacZ activity points represent the averages of three separate cultures
done in duplicate. Individual specific activities never varied by more
than 15%.
Figure 5:
Effect of carbon source on YHG expression.
YM4134 cells were grown in YP medium supplemented with 2% glucose, 2%
galactose, 2% raffinose, 4% glycerol, 4% lactate, or 4% ethanol. The
latter three cultures were also supplemented with 0.1% galactose for
enhanced early growth. Total RNA was isolated from midlog phase
cultures, and the subsequent blot was hybridized with YHG. ACT1 probe
was used as an mRNA loading standard.
Figure 6:
Northern and Western analyses of YHG
knockout. A, total RNA was isolated and blotted from YM4134 (wt) and the hemoglobin knockout strain YD7 (-yhg). The membrane was hybridized to YHG probe and,
after stripping, with ACT1 probe. B, total protein from 1.5
No phenotypic differences were found between the
wild-type and the yhg strains in a variety of conditions
tested, including growth on fermentable or non-fermentable carbon
sources from 100% oxygen to strict anaerobiosis. When induced to lose
its mitochondrial DNA by ethidium bromide treatment(46) , the
knockout strain formed petite colonies with no effect on fermentative
growth (not shown). Regulation of the S. cerevisiae flavohemoglobin gene
is clearly controlled by cell density and oxygen tension but in a
manner different from that of the bacterial globins previously studied.
During our efforts to correlate YHG transcriptional activation with
oxygen concentration, we were able to determine at what oxygen tension
the apparent switchover from aerobic to anaerobic gene expression in S. cerevisiae occurs. The messages for both the aerobic TIF51A
and the anaerobic TIF51B are found at 0.1% oxygen. Above this
concentration, only TIF51A is present; below this concentration, only
TIF51B can be detected. A similar result was seen for the COX5a/b gene
pair. This is also the oxygen tension where aerobic activation of the
YHG promoter ends and the anaerobic expression of the YHG message
begins. Thus, 0.1% likely straddles the concentration of oxygen
necessary for heme biosynthesis. Aerobic expression of YHG is
predominantly activated by the HAP1 and HAP2/3/4 transcription factor
complexes. An anaerobic system to produce YHG message independent of
the HAPs is also present. Although there is a decrease in YHG gene
expression as the cells approach anaerobiosis, the effect is not as
dramatic as the depletion in YHG mRNA as the cells reach higher optical
densities ( Fig. 1compared with Fig. 2). The additional
repression of endogenous YHG gene expression upon exit from exponential
phase may be due to a combination of factors such as hypoxic
conditions, induction of a repressor, or the altered chromatin
structure at higher culture densities that has been implicated in
repressing most exponential phase mRNA species(47) . Our
results indicate that the YHG promoter/lacZ fusion construct
is also regulated by heme and the HAPs, but interesting quantitative
differences between Northern and transcriptional fusion analyses exist.
The hem1 and hap1/hap2 strains display 4- and 10-fold
decreases in lacZ activity relative to wild type, whereas
quantitation of Northern analyses indicate a YHG mRNA decrease of 8-
and 50-fold for the hem1 and hap1/hap2 strains,
respectively ( Fig. 3and Table 2). The drop in lacZ activity as the cells exit log phase is also less dramatic than
that of the native YHG message ( Fig. 4compared with Fig. 1). These results raise the possibility that sequences
outside of the 1.6-kb upstream region governing the lacZ fusion influence transcription or stability of the endogenous YHG
gene product. The physiological function of hemoglobins in species
other than vertebrate animals has not been well established. The
present data suggest that, despite the high degree of homology between
globins of prokaryotic and eukaryotic microorganisms, each species may
have developed a discrete role for its hemoglobin. Through activation
by bacterial FNR or related transcription factors, hemoglobins of the
strictly respiring Vitreoscilla and A. eutrophus species are probably needed for metabolism when oxygen supplies
are severely limiting or absent(9, 15) .
Overexpression studies in E. coli indicate that the Vitreoscilla globin can increase oxygen uptake and can even
act as a terminal oxidase(4, 5) . An A. eutrophus strain carrying a deletion of the flavohemoglobin gene could not
transiently accumulate nitrous oxide during anaerobic growth using
nitrate as the terminal electron acceptor, suggesting a role of this
protein in the metabolism of a gas other than dioxygen(9) . A
specialized role for the A. eutrophus globin is suggested by
the positioning of the native flavohemoglobin gene on an episomal
plasmid linked to denitrification processes (48) and by the
inability of species that contain homologous globins, such as S.
cerevisiae, to use nitrite as an anaerobic terminal electron
acceptor(49) . Another area of variability between members of
the flavohemoglobin family is in their affinities for oxygen. The
dissociation constant for the C. norvegensis flavohemoglobin
in solution is 2 Clues to the possible
function of the S. cerevisiae flavohemoglobin have been
uncovered by the gene regulation results presented here. Unlike the Vitreoscilla globin, YHG is probably not involved in
facilitating oxygen storage or diffusion during hypoxic electron
transport. Contrary to many other genes involved in respiration, the
YHG message represents the first known example of a HAP2/3/4-regulated
gene that is not glucose-repressed, indicating that the gene may be
required in both fermentable and non-fermentable carbon sources. There
is an increase in YHG mRNA levels in cells grown under high oxygen
tension. This phenomenon can be attributed to increased heme levels,
which also stimulate superoxide dismutase and catalase transcription in S. cerevisiae(53, 54) . Flavohemoglobin in S. cerevisiae may also be involved in the detoxification of
oxygen. It should be noted, however, that no detectable growth
difference is seen between wild-type or yhg strains when grown
in 100% oxygen or in the presence of hydrogen peroxide (not shown). The capacity of the YHG promoter to remain active even during
anaerobic growth suggests that the hemoglobin may be necessary in
certain anaerobic conditions. A phenotype for the A. eutrophus strain lacking hemoglobin was found only when cells were grown
anaerobically(9) . It can be postulated that low levels of YHG
might be needed anaerobically to quickly initiate a signal cascade once
oxygen becomes available. Activity of the C-terminal reductase domain
in YHG could be altered with the binding of heme or heme and oxygen in
the N-terminal portion of the molecule. A heme-containing oxygen sensor
has been found in the Rhizobium bacteria, where an
oxygen-regulated kinase in the N terminus is linked to a heme-binding
domain in the C terminus(55) . Biochemical studies of the E. coli flavohemoglobin indicate that flavin reduction in the
protein is increased upon deoxygenation, suggesting that the reductase
activity may be altered by the availability of oxygen(52) .
Further phenotypic studies of the YHG knockout strain and biochemical
analyses of the YHG protein should provide insight into the role of
flavohemoglobins from facultative eukaryotic microorganisms.
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6991-6996
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)allows increased cell densities and
rescues terminal oxidase mutants in Escherichia coli,
indicating that VtHb is capable not only of delivering molecular oxygen
but also of facilitating its reduction in
vivo(4, 5) . Hemoglobins recently sequenced from
the bacteria E. coli and Alcaligenes eutrophus and
the yeasts Saccharomyces cerevisiae and Candida
norvegensis are two-domain proteins with nearly 40% sequence
identity(6, 7, 8, 9) . Their
N-terminal regions share substantial sequence homology with the
single-domain VtHb, whereas the C terminus contains a reductase domain
with potential binding sites for flavin (FAD) and NADPH. Because of the
additional domain, this class of globins has also been called
flavohemoglobins. The native VtHb interacts with a separate reductase,
making it a two-polypeptide analog of the
flavohemoglobins(10) .
Strains and Plasmids Used
The S. cerevisiae strains used in this study are listed in Table 1. To
facilitate deletion of the yeast hemoglobin gene, 3` portions of the S. cerevisiae flavohemoglobin (YHG) coding region were fused
upstream of 5` portions (22) by polymerase chain reaction
(PCR). A unique BamHI restriction site, necessary for the
subsequent transformation in yeast, was incorporated into the joint
between gene fragments. The fused sequence was subcloned into plasmid
pRS305 (Leu selection), and the S. cerevisiae hemoglobin-deletion strain YD7 was constructed by
-recombination into YM4134(23) .
1.6 kb
of YHG promoter into the SpeI site of the lacZ expression plasmid yIP357R(23) . Standard techniques in
the manipulation of these shuttle vectors and propagation of E.
coli were used(24) . Yeast transformations were performed
using the LiAc method as described (25) and plated onto
selective media(26) .Media and Growth Conditions
For RNA analyses of
growth phase, oxygen curve, and heme/transcription factor-related
expression, cultures were grown in YPGal media (1% yeast extract, 2%
Bactopeptone, 2% galactose) at 30 °C. Media components were from
Difco or Sigma. The hem1 strain was supplemented with 10
µg/ml ergosterol with 0.1% Tween 80 and/or 100 µg/ml
-aminolevulinic acid. For analysis of carbon source regulation,
cultures were isolated in midlog phase grown in YP media with 2%
glucose, galactose, or raffinose. Cultures grown in glycerol, lactate,
and ethanol were supplemented with 4% carbon source and 0.1% galactose.
For growth at various oxygen tensions, a small inoculum of YM4134
overnight culture was placed in a 250-ml side-arm flask containing 100
ml of YPGal supplemented with 10 µg/ml ergosterol and 0.1% Tween
80. Cultures were immediately placed in a closed system wherein defined
oxygen concentrations (from 100 to 0%, balanced with nitrogen) were
bubbled from tanks (Genex) through the media. The oxygen tensions below
0.03% were monitored by using an anaerobic indicator strip (BBL
Microbiology Systems). Cultures were grown for at least 12 h at 30
°C with shaking. Growth was monitored by a Klett meter until midlog
phase was reached. Then the flasks, still subjected to the flow of gas,
were placed on ice for 20 min. 5-ml aliquots were subsequently removed
for RNA extraction.Extraction and Analysis of RNA
RNA was isolated
from cells using the glass bead method in the presence of
phenol/chloroform(27) . 10 µg of total RNA was loaded onto
1.2% agarose gels containing 3% formaldehyde. After electrophoresis,
gels were ethidium-stained to monitor RNA integrity and then
transferred to a Magnagraph nylon membrane (Micron Separations, Inc.).
To ensure equal loading of mRNA, the membrane was hybridized to a probe
made from the S. cerevisiae actin (ACT1) gene. All probe
templates were created by polymerase chain reaction using the following
primers based on the published sequences of YHG, ACT1, TIF51B, and
COX5(8, 28, 29, 30) : YHG 5`,
5`-ATGCTAGCCGAAAAAACCC-3`; YHG 3`, 5`-CTAAACTTGCACGGTTGAC-3`; ACT1 5`,
5`-GGTTGCTGCTTTGGTTATTG-3`; ACT1 3`, 5`-TTAGAAACACTTGTGGTGAAC-3`; TIF51
5`, 5-ATGTCTGACGAAGAACACAC3`; TIF51 3`, 5`-CTAATCAGATCTTGGAGCTT-3`;
COX5 5`, 5-AAGATT(T/C)G(T/C)TCAAACA(A/C)A(G/T)GC(T/C)CTTTC-3`; COX5 3`,
5`-T(T/C)ATTTAGATTG(G/A)AC(T/C)TGAGAATAACC(A/T)CC-3`. All genes were
amplified by PCR from genomic DNA of YM4134. The products, which were
of predicted size, were subsequently used as a template for probe
synthesis by PCR incorporation of
[
-
P]dATP(31) . Exposed films were
assayed for band intensity using a scanning laser densitometer
(Molecular Dynamics).Measurement of
All
strains described were transformed with the integrating plasmid pYH10
containing the wild-type hemoglobin promoter driving the lacZ gene (32) and selected on minimal media (-ura). Two
independent transformants were used for further study.
-Galactosidase Activity-Galactosidase assays (33) were performed in duplicate on
midlog-phase cells permeabilized with SDS and chloroform. The specific
activity is expressed as (A
3500)/(min A
of cells).Physical and Genetic Mapping
The physical map
location of YHG was determined using 
clone grid filters obtained
from Linda Riles of Washington University(34) . The filters
were used as described in the protocol accompanying the filter set.
Genetic crosses between YD7 and yJC366 and subsequent sporulation and
tetrad dissection of YGGM1 were performed using standard
techniques(35) .YHG Antibody Production and Western Blotting
The
YHG gene was overexpressed in E. coli using the PET vector
system (36) . After partial purification(8) , gel
slices of the 47-kDa protein, confirmed as YHG by N-terminal amino acid
sequencing, were used for rabbit immunization(37) . For Western
analysis, total S. cerevisiae protein was fractionated by
SDS-polyacrylamide gel electrophoresis(38) , transferred to
nitrocellulose, incubated with primary antiserum (1:100) followed by
alkaline phosphatase-conjugated secondary antibody (Bio-Rad), and
developed as described(39) .
Hemoglobin mRNA Levels Decrease as the Cultures Exit
Log Phase
Vitreoscilla and A. eutrophus hemoglobin levels increase as nutrient and oxygen concentrations
become limiting when the cells exit the logarithmic growth
period(11, 14) . S. cerevisiae also possesses
gene products involved in oxygen metabolism whose levels increase after
the cessation of log phase. These include the cytoplasmic catalase (CTT1) gene and the CYC7 gene, encoding iso-2
cytochrome c(40, 41) . To determine whether
the S. cerevisiae hemoglobin follows this pattern, we
inoculated a dilute sample of YM4134 into YPGal media and removed
aliquots for steady-state mRNA isolation at the cell densities
indicated in Fig. 1. The log-phase doubling time of 2 h in YPGal
for this strain ceased at an A
of about 2.5.
Stationary phase, determined by the lack of any increase in A after 48 h of continuous shaking at 30 °C,
was reached at an A of about 10.5. To be sure
that cells were still viable, cultures were plated on solid YPD media,
and subsequent colony counts were found to correlate well with cell
densities. As shown in Fig. 1, the YHG message begins to
decrease as the cells exit log phase and appears to be absent from
stationary phase cultures. Densitometry analyses indicate a 25-fold
decrease in YHG mRNA by an A of 5.5 and greater
than a 100-fold decrease at higher optical densities. Levels of YHG
mRNA also drop sharply upon exit from log phase when synthetic media or
a non-fermentable carbon source is used (not shown), suggesting a
global repression independent of nutrient source. The decrease in
hemoglobin message upon exit from log-phase growth indicates that the
hemoglobin is probably not required for oxygen-depleted respiration
after the diauxic shift from log-phase growth in S.
cerevisiae.
cells. Cultures
were grown in YPGal media with vigorous shaking at 30 °C. At the
points indicated by the arrows, aliquots of culture were
removed and total RNA isolated. The numbers above the inset indicate the A at the time of RNA
isolation. 10 µg of total RNA was loaded into each lane.
The RNA blot was probed with a radiolabeled YHG PCR product as
described under ``Materials and Methods.'' Densitometry
readings of the band intensities (setting A of
1.5 equal to 1.0) were, in order: 1.0, 1.0, 0.78, 0.69, 0.04, <0.01,
and <0.01. Ethidium-stained intensity of the 18 S rRNA band was used
as an internal RNA loading standard and gave intensities: 1.0, 1.0,
1.0, 1.2, 1.2, 0.93, and 0.81.
Effect of Oxygen Tension on the Transcription of
YHG
The depletion of hemoglobin message as the cells exit log
phase raises the possibility that intracellular oxygen levels may play
a role in the expression of YHG. To examine this, we set up a
variable-oxygen cell growth system. Cultures of YM4134 were inoculated
into YPGal and continuously purged with defined gas mixtures (ranging
from 100% O
to 0% O/100% N) until
midlog phase was reached (A of 1-1.5). As
shown in Fig. 2, the hemoglobin message is maximal in 100%
oxygen and decreases as oxygen levels decline to about 0.1%. Compared
with 21% O by densitometry, a 4-fold lower but constant
amount of message is detected from 0.1% oxygen down to strict
anaerobiosis. Hemoglobin expression as a function of oxygen
concentration was compared with the well characterized,
oxygen-dependent message for translation initiation factor eIF-5a. In S. cerevisiae, eIF-5a is encoded by the aerobic/anaerobic
gene pair TIF51A and TIF51B (also known as ANB1), which share 90%
nucleotide sequence identity(42) . Fig. 2indicates that
the switch from aerobic to anaerobic conditions for TIF51A and TIF51B
occurs at about 0.1% oxygen, where both are similar in their
expression. Above 0.1% O no TIF51B mRNA is detected, and
below 0.1% O no TIF51A message is seen. A similar pattern
was found for the COX5 aerobic/anaerobic gene pair when this blot was
analyzed with a COX5-specific probe (not shown).
Deficiency of Heme and Heme-induced Transcription Factors
Decrease Aerobic YHG Expression
A key intermediate in the
pathway from oxygen levels to regulation of gene expression is heme,
the biosynthesis of which requires molecular oxygen at two
steps(19) . S. cerevisiae mutants that cannot
synthesize heme mimic an oxygen-depleted state, repressing the
expression of aerobic genes while activating anaerobic
genes(16, 17) . The only known direct link between
heme and oxygen-regulated gene expression is the interaction of heme
with HAP1, a transcription factor that activates a variety of aerobic
genes(16, 17) . Heme appears to function by
facilitating the formation of an active HAP1/DNA complex or by altering
the complement of proteins assembled at a HAP1-regulated
promoter(41, 42) . The pathway leading to activation
by HAP2/3/4, a heterotrimeric transcription factor complex involved in
heme-induced transcription of CYC1, cytochrome oxidase subunit
5a (COX5a), and other aerobic genes, is currently
unclear(16, 18) . Null alleles of the HAP2 polypeptide
are sufficient to prevent complex assembly and subsequent activation at
a HAP2/3/4-dependent promoter(43) .-aminolevulinic
acid synthase, the enzyme catalyzing the first step in heme
biosynthesis(19) . As shown in Fig. 3A, heme
deficiency results in significantly reduced amounts of YHG mRNA (about
8-fold by densitometry). Levels return to those of wild type with the
addition of 100 µg/ml -aminolevulinic acid, which circumvents
the hem1 lesion in the pathway.
-aminolevulinic acid (hem1 + ALA). Setting wild type
as 1.0 for band intensity, the following densitometry results were
obtained: 1.0, 0.12, and 1.41. B, total RNA was isolated from
strains BWG7-1a (wt), LPY22 (hap1),
JO1-1a (hap2), and JS109 (hap1/hap2) grown
under aerobic and anaerobic (N) conditions. Both parts of
the experiment are from the same blot, and the N lanes have
been placed underneath the aerobic lanes for comparison. Both aerobic
and anaerobic conditions produce the identical 1.2-kb YHG mRNA. The
ACT1 probe was used as an internal standard for mRNA loading.
Densitometry results for the YHG band (with wild type in air set at
1.0) are: 1.0, 0.46, 0.29, and 0.02 in air and 0.25, 0.10, 0.10, and
0.11 in nitrogen.
-galactosidase produced by the wild-type strain. The YHG
promoter/lacZ fusion construct integrated into the wild-type
strain YM4134 also displayed a 3-fold decrease in YHG promoter activity
as the cells exited log phase (Fig. 4) or were subjected to
decreased oxygen tensions (not shown).
Expression of Hemoglobin Is Independent of Carbon
Source
All of the genes regulated by the HAP2/3/4 complex
studied to date are subject to transcriptional repression in the
presence of glucose, including those that are dually activated by
HAP1(16, 18) . S. cerevisiae will
preferentially ferment glucose even in the presence of oxygen, and many
of the genes involved in utilization of other carbon sources are
repressed by an order of magnitude or more when cells are grown in
glucose-containing media(44) . Induction of HAP2/3/4-dependent
promoters in media containing carbon sources other than glucose appears
to be correlated with activation of HAP4 transcription(44) . To
determine if the S. cerevisiae hemoglobin expression is
glucose-repressed, cells were grown in media containing a variety of
carbon sources. As shown in Fig. 5, no significant induction
over glucose mRNA levels could be found with cells grown in galactose,
raffinose, or the non-fermentable glycerol, lactate, and ethanol carbon
sources. By both Northern analyses and hemoglobin promoter/lacZ activity assays (not shown), a similar lack of glucose repression
in the YHG promoter was observed in synthetic media supplemented with
various carbon sources.
Physical and Genetic Map Location of
Flavohemoglobin
We physically mapped the YHG locus to determine
if any genetic markers related to oxygen metabolism were in the region.
The YHG PCR probe was hybridized to a set of clones that cover
over 90% of the yeast genome(34) . The results identified a
clone that contains a fragment of the right arm of chromosome VII
within 40 kb of ADE3. A respiration-deficient mutant (PET54)
is also in this region, encoding a previously described mitochondrial
translational factor(45) . For genetic mapping and subsequent
phenotypic characterization, the LEU2 gene was inserted into
the hemoglobin locus by -recombination(23) . A
site-specific disruption of the YHG locus was confirmed by Southern
blot (not shown) as well as Northern and Western analyses (Fig. 6). The knockout strain, YD7, was crossed to yJC366, a
strain containing a mutation in ADE3. The yhg locus
was followed by scoring the LEU2 marker, and the ADE3 marker was followed by scoring red colonies(35) . Genetic
mapping confirmed the presence of the flavohemoglobin locus on the
right arm of chromosome seven, 15.4 centimorgans (19PD: 0NPD:8TT) from ADE3.
10
log-phase cells of YM4134 and YD7 was run on
SDS-polyacrylamide gel electrophoresis and probed with rabbit -YHG
antiserum raised against recombinant YHG expressed in E. coli.
Numbers corresponding to the 52- and 34-kDa molecular mass markers are
shown. The 47-kDa band seen in the wt lane is the predicted
size of the YHG protein(8) .
10
M, making it
one of the most oxygen-avid hemoglobins known(50) . The
dissociation constants for the Vitreoscilla and E. coli hemoglobins are 7.2 10
and 2
10
M, respectively, a difference of at
least 2 orders of magnitude from the C. norvegensis globin,
despite nearly 50% sequence identity in the heme-binding domains of
these proteins(51, 52) .
)
We would like to thank John Cooper, Leonard Guarente,
and Richard Zitomer for the provision of strains and John Lawrence for
use of the densitometer. We are indebted to Mark Johnston, Linda Riles,
and Tina Hesman for the provision of yeast strains as well as for
invaluable discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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